Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

PubMed

Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

2015-06-01

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

Crossing borders to bind proteins--a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set.

PubMed

Baltzer, Lars

2011-06-01

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

DOEpatents

Freimuth, Paul I.

2010-04-06

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

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.

[beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose.

PubMed Central

Li, L.; Drake, R. R.; Clement, S.; Brown, R. M.

1993-01-01

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed. PMID:12231766

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.

PubMed

Montgomery, H J; Romanov, V; Guillemette, J G

2000-02-18

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

PubMed Central

1986-01-01

A collection of 17 monoclonal antibodies elicited against the light- harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC- II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b. PMID:3528171

Leiurotoxin I (scyllatoxin), a peptide ligand for Ca2(+)-activated K+ channels. Chemical synthesis, radiolabeling, and receptor characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auguste, P.; Hugues, M.; Grave, B.

1990-03-15

Leiurotoxin I (scyllatoxin) is a 31-amino acid polypeptide from the venom of the scorpion Leiurus quinquestriatus hebraeus which has been previously isolated and sequenced by others. This paper reports (i) the total synthesis of this scorpion neurotoxin as well as some aspects of its structure-function relationships; (ii) the synthesis of the analog (Tyr2)leiurotoxin I (scyllatoxin) that has been monoiodinated at high specific radioactivity (2000 Ci/mmol) and has served for the characterization of the properties of {sup 125}I-(Tyr2)leiurotoxin I binding sites (Kd = 80 pM, molecular mass of 27 and 57 kDa for two polypeptides in the leiurotoxin I binding protein);more » (iii) the similarity of physiological actions between leiurotoxin I and apamin. Both toxins contract Taenia coli previously relaxed with epinephrine, both toxins block the after-hyperpolarization due to Ca2(+)-activated K+ channel activity in muscle cells in culture; (iv) the probable identity of binding sites for apamin and leiurotoxin I. In spite of a different chemical structure apamin competitively inhibits {sup 125}I-(Tyr2) leiurotoxin I binding and vice versa. Moreover, the peculiar effects of K+ on {sup 125}I-(Tyr2)leiurotoxin I binding are identical to those previously observed for {sup 125}I-apamin binding.« less

The two subunits of the human asialoglycoprotein receptor have different fates when expressed alone in fibroblasts

PubMed Central

Shia, Michael A.; Lodish, Harvey F.

1989-01-01

Two related polypeptides, H1 and H2, comprise the human asialoglycoprotein receptor (ASGP-R). Stable lines of murine NIH 3T3 fibroblasts expressing H1 alone or H2 alone do not bind or internalize the ligand asialoorosomucoid (ASOR), which contains triantennary oligosaccharides. In contrast, cells expressing H1 and H2 together bind and degrade ASOR with properties indistinguishable from those of the ASPG-R in human hepatoma HepG2 cells. Whether or not H2 is coexpressed, H1 is synthesized as a 40-kDa precursor bearing high-mannose oligosaccharides, processed to its mature 46-kDa form, and transported to the cell surface. In cells expressing only H1, homodimers and -trimers of H1 are formed. In contrast, when expressed in 3T3 cells without H1, H2 is synthesized as its 43-kDa precursor, bearing high-mannose oligosaccharides, but is rapidly degraded. When H1 and H2 are coexpressed in the same cell, the H1 polypeptide “rescues” the H2 polypeptide; H2 is processed to its characteristic 50-kDa mature form and is transported to the surface. We conclude that the human ASGP-R is a multichain heterooligomer, probably a trimer of H1 molecules in noncovalent association with one, two, or three H2 molecules, and that the two polypeptides normally interact early in biosynthesis. Images PMID:2919187

Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain.

PubMed

Catterall, W A; Hartshorne, R P; Beneski, D A

1982-01-01

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

PubMed

Badache, A; Burger, D; Villarroya, H; Robert, Y; Kuchler, S; Steck, A J; Zanetta, J P

1992-01-01

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of the heterogeneous subunits of elongation factor EF-1 from Guerin epithelioma cells.

PubMed

Marcinkiewicz, C; Gajko, A; Gałasiński, W

1991-01-01

Elongation factor EF-1 from Guerin epithelioma was separated into two subunit forms EF-1A and EF-1B by chromatography in the presence of 25% glycerol, successively on CM-Sephadex and DEAE-Sephadex. It was shown that EF-1A is a thermolabile, single polypeptide which catalyses the binding of aminoacyl-tRNA to ribosomes, similarly as eukaryotic EF-1 alpha or prokaryotic EF-Tu. EF-1B was characterized as a complex composed of at least two polypeptides. One of them is EF-1A, the other EF-1C, which stimulates EF-1A activity and protects this elongation factor from thermal inactivation.

Engineering nanomaterials with a combined electrochemical and molecular biomimetic approach

NASA Astrophysics Data System (ADS)

Dai, Haixia

Biocomposite materials, such as bones, teeth, and shells, are created using mild aqueous solution-based processes near room temperature. Proteins add flexibility to these processes by facilitating the nucleation, growth, and ordering of specific inorganic materials into hierarchical structures. We aim to develop a biomimetic strategy for engineering technologically relevant inorganic materials with controlled compositions and structures, as Nature does, using proteins to orchestrate material formation and assembly. This approach involves three basic steps: (i) preparation of inorganic substrates compatible with combinatorial polypeptide screening; (ii) identification of inorganic-binding polypeptides and their engineering into inorganic-binding proteins; and (iii) protein-mediated inorganic nucleation and organization. Cuprous oxide (Cu2O), a p-type semiconductor, has been used to demonstrate all three steps. Zinc oxide (ZnO), an n-type semiconductor, has been used to show the generality of selected steps. Step (i), preparation of high quality inorganic substrates to select inorganic-binding polypeptides, was accomplished using electrochemical microfabrication to grow and pattern Cu2O and ZnO. Raman spectroscopy and x-ray photoelectron spectroscopy were used to verify phase purity and compositional stability of these surfaces during polypeptide screening. Step (ii), accomplished in collaboration with personnel in Prof Baneyx' lab at the University of Washington, involved incubating the inorganic substrates with the FliTrx(TM) random peptide library to identify cysteine-constrained dodecapeptides that bind the targeted inorganic. Insertion of a Cu2O-binding dodecapeptide into the DNA-binding protein TraI endowed the engineered TraI with strong affinity for Cu2O (Kd ≈ 10 -8 M). Finally, step (iii) involved nonequilibrium synthesis and organization of Cu2O nanoparticles, taking advantage of the inorganic and DNA recognition properties of the engineered TraI. The high affinity of the engineered TraI for Cu2O over other related copper compounds led to the formation of Cu2O nanoparticles from a cuprous chloride complex (Cu2Cln1-n, n = 2 or 3) electrolyte under conditions where the mineral atacamite (CuCl(OH) 3) is thermodynamically preferred. The nonequilibrium Cu 2O nanoparticles consisted of 2--3 nm Cu2O cores and functional protein shells that enabled predictable meso-scale assembly on DNA templates. In short, we have rationally designed a protein-based scheme for forming and organizing inorganic materials that Nature has not previous worked with.

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.

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harada, Y.; Li, H.; Li, Hua

2009-04-28

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings,more » we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.« less

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

PubMed Central

Harada, Yoichiro; Li, Hua; Li, Huilin; Lennarz, William J.

2009-01-01

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of ≈1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes. PMID:19365066

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.

Profiling the Serum Protein Corona of Fibrillar Human Islet Amyloid Polypeptide.

PubMed

Pilkington, Emily H; Gustafsson, Ove J R; Xing, Yanting; Hernandez-Fernaud, Juan; Zampronio, Cleidi; Kakinen, Aleksandr; Faridi, Ava; Ding, Feng; Wilson, Paul; Ke, Pu Chun; Davis, Thomas P

2018-05-16

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona in vivo that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis, we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in fetal bovine serum using two complementary methodologies developed herein: quartz crystal microbalance and "centrifugal capture", coupled with nanoliquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, whereas strong binding with IAPP fibrils occurred for linear proteins or multidomain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets.

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

PubMed Central

Pollock, W B; Loutfi, M; Bruschi, M; Rapp-Giles, B J; Wall, J D; Voordouw, G

1991-01-01

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes. Images PMID:1846136

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

PubMed

Mossabeb, Roschanak; Seiberler, Susanne; Mittermann, Irene; Reininger, Renate; Spitzauer, Susanne; Natter, Susanne; Verdino, Petra; Keller, Walter; Kraft, Dietrich; Valenta, Rudolf

2002-10-01

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.

Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers.

PubMed

Negri, Graciela E; Deming, Timothy J

2017-01-01

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting.

PubMed

Swainsbury, David J K; Martin, Elizabeth C; Vasilev, Cvetelin; Parkes-Loach, Pamela S; Loach, Paul A; Neil Hunter, C

2017-11-01

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880nm to 915nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, F.C.; Brown, R.M. Jr.; Drake, R.R. Jr.

1990-03-25

Photoaffinity labeling of purified cellulose synthase with (beta-32P)5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of (beta-32P)5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin.

PubMed

Hou, Hu; Li, Bafang; Zhang, Zhaohui; Xue, Changhu; Yu, Guangli; Wang, Jingfeng; Bao, Yuming; Bu, Lin; Sun, Jiang; Peng, Zhe; Su, Shiwei

2012-12-01

Collagen polypeptides were prepared from cod skin. Moisture absorption and retention properties of collagen polypeptides were determined at different relative humidities. In addition, the protective effects of collagen polypeptide against UV-induced damage to mouse skin were evaluated. Collagen polypeptides had good moisture absorption and retention properties and could alleviate the damage induced by UV radiation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing the endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

PubMed

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

The Urtica dioica Agglutinin Is a Complex Mixture of Isolectins 1

PubMed Central

Van Damme, Els J. M.; Broekaert, Willem F.; Peumans, Willy J.

1988-01-01

Rhizomes of stinging nettle (Urtica dioica) contain a complex mixture of isolectins. Ion exchange chromatography with a high resolution fast protein liquid chromatography system revealed six isoforms which exhibit identical agglutination properties and carbohydrate-binding specificity and in addition have the same molecular structure and virtually identical biochemical properties. However, since the U. dioica agglutinin isolectins differ definitely with respect to their amino acid composition, it is likely that at least some of them are different polypeptides coded for by different genes. Images Fig. 3 PMID:16665952

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Functional domains of the T lymphocyte plasma membrane: characterization of the polypeptide composition.

PubMed

Szamel, M; Kaever, V; Resch, K

1987-01-01

Highly purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on Concanavalin A-Sepharose into two subfractions, one eluting freely from the affinity column (MF1) and a second being specifically retained (MF2). SDS-polyacrylamide-gel-electrophoresis revealed different polypeptide patterns of the two plasma membrane subfractions. Polypeptides of apparent molecular weights of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2. In contrast, several proteins in the 55-65 kDa range were preferentially recovered in the non-adherent fraction. Five Five of the six polypeptides, preferentially recovered in MF2 proved to be glycoproteins, the 39 kDa peptide was non-glycosilated. The differences in the amounts of the polypeptides specifically enriched in the adherent fraction MF2 became even more clear-cut when plasma membranes solubilized with non-ionic detergents (lysolecithin, ET-18-2H, Triton-X-100) were separated by affinity chromatography on Concanavalin A-Sepharose. The non-glycosilated peptide of apparent molecular weight of 39 kDa was recovered together with several glycoproteins in the adherent fraction, MF2, suggesting that not single glycoproteins, but plasma membrane domains were separated by Concanavalin A-Sepharose. Although the glycoproteins of the non-adherent fraction MF1 bound significant amounts of Concanavalin A, the major Concanavalin A binding glycoproteins were recovered in the adherent fraction, MF2. The plasma membrane subfractions showed also different functional properties, the specific activities [Na+ + K+]AT-Pase, Ca2+ ATPase and lysolecithin acyltransferase were several-fold enriched in the adherent fraction, MF2, as compared to MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of thymocytes consisting of a different set of proteins, among others the major Concanavalin A binding glycoproteins with some membrane bound enzymes, probably implicated in the initiation of lymphocyte activation.

Recombinant soluble adenovirus receptor

DOEpatents

Freimuth, Paul I.

2002-01-01

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

Direct DNA binding by Brca1.

PubMed

Paull, T T; Cortez, D; Bowers, B; Elledge, S J; Gellert, M

2001-05-22

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide

PubMed Central

Singh, Pradeep K.; Ghosh, Dhiman; Tewari, Debanjan; Mohite, Ganesh M.; Carvalho, Edmund; Jha, Narendra Nath; Jacob, Reeba S.; Sahay, Shruti; Banerjee, Rinti; Bera, Amal K.; Maji, Samir K.

2015-01-01

Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail. PMID:25803428

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

PubMed

Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

2016-03-01

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome reaction, whereas the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent-soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction.

Purification and characterization of human mitochondrial transcription factor 1.

PubMed Central

Fisher, R P; Clayton, D A

1988-01-01

We purified to near homogeneity a transcription factor from human KB cell mitochondria. This factor, designated mitochondrial transcription factor 1 (mtTF1), is required for the in vitro recognition of both major promoters of human mitochondrial DNA by the homologous mitochondrial RNA polymerase. Furthermore, it has been shown to bind upstream regulatory elements of the two major promoters. After separation from RNA polymerase by phosphocellulose chromatography, mtTF1 was chromatographed on a MonoQ anion-exchange fast-performance liquid chromatography column. Analysis of mtTF1-containing fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single major polypeptide with an Mr of approximately 25,000. Centrifugation in analytical glycerol gradients indicated a sedimentation coefficient of approximately 2.5 S, consistent with a monomeric 25-kilodalton protein. Finally, when the 25-kilodalton polypeptide was excised from a stained sodium dodecyl sulfate-polyacrylamide gel and allowed to renature, it regained DNA-binding and transcriptional stimulatory activities at both promoters. Although mtTF1 is the only mitochondrial DNA-binding transcription factor to be purified and characterized, its properties, such as a high affinity for random DNA and a weak specificity for one of its target sequences, may typify this class of regulatory proteins. Images PMID:3211148

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.

PubMed

Kochibe, N; Matta, K L

1989-01-05

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.

Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site

PubMed Central

Song, Lingyun; Caguiat, Jonathan; Li, Zhongrui; Shokes, Jacob; Scott, Robert A.; Olliff, Lynda; Summers, Anne O.

2004-01-01

The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step. PMID:14996817

Serological Reactivity and Identification of IgE-Binding Polypeptides of Ganoderma applanatum Crude Spore Cytoplasmic Extract in Puerto Rican Subjects

PubMed Central

Vilá-Héreter, Frances; Rivera-Mariani, Felix E.; Bolaños-Rosero, Benjamín

2017-01-01

Background The allergenic potential of Ganoderma applanatum basidiospores has been demonstrated previously in Puerto Rico. However, basidiomycete allergens are not available for inclusion in allergy diagnostic panels. Therefore, we sought to confirm allergic sensitization towards G. applanatum crude spore cytoplasmic extract (CSCE) through reactivity in serological assays and detection of IgE-binding polypeptides. Methods With an indirect ELISA, serological reactivity was compared between groups of individuals with different allergic profiles. Group 1 (n = 51) consisted of individuals with sIgE to allergens included in diagnostic panels; group 2 (n = 14) were individuals with no sIgE to the allergens tested; and group 3 (n = 22) were individuals with no allergic history. To visualize IgE-binding polypeptides, group 1 sera were examined with Western blot (WB). Polypeptide bands with the highest reactivity were analyzed by mass spectrometry (MS) for putative identification. Results Serological reactivity of group 1 was significantly higher than that of group 3 in indirect ELISA (p = 0.03). Sixty five percent of group 1 individuals showed reactivity to polypeptide bands in WB. Bands of 81 and 56 kDa had the highest reactivity proportions among the reactive sera, followed by a 45 kDa band. MS analysis of these three polypeptides suggests they are basidiomycete-derived enzymes with aconitate hydratase, catalase, and enolase functions. Conclusions G. applanatum spores have allergenic components recognized by Puerto Rican individuals, which could eventually be considered as markers in cases of fungal allergy and be included in diagnostic allergen panels in Puerto Rico and tropical regions. PMID:28380479

Double-hydrophobic elastin-like polypeptides with added functional motifs: Self-assembly and cytocompatibility.

PubMed

Le, Duc H T; Tsutsui, Yoko; Sugawara-Narutaki, Ayae; Yukawa, Hiroshi; Baba, Yoshinobu; Ohtsuki, Chikara

2017-09-01

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and β-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017. © 2017 Wiley Periodicals, Inc.

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

Identification of Peroxiredoxin-5 in Bovine Cauda Epididymal Sperm

PubMed Central

Nagdas, Subir K; Buchanan, Teresa; Raychoudhury, Samir

2013-01-01

Developing spermatozoa require a series of post-testicular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5kDa Wheat Germ Agglutinin (WGA) binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA stained bands, the presence of a 17.5kDa WGA binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide. PMID:24186847

IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Victoria, E.J.; Pierce, S.W.; Branks, M.J.

1990-01-01

Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting frommore » the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10%, consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3.« less

Identification of the triazine receptor protein as a chloroplast gene product

PubMed Central

Steinback, Katherine E.; McIntosh, Lee; Bogorad, Lawrence; Arntzen, Charles J.

1981-01-01

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed “B.” The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[14C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [35S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[14C]atrazine, [35S]methionine in vivo, or [35S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. Images PMID:16593133

Serological Reactivity and Identification of Immunoglobulin E-Binding Polypeptides of Ganoderma applanatum Crude Spore Cytoplasmic Extract in Puerto Rican Subjects.

PubMed

Vilá-Héreter, Frances; Rivera-Mariani, Félix E; Bolaños-Rosero, Benjamín

2017-01-01

The allergenic potential of Ganoderma applanatum basidiospores has been demonstrated previously in Puerto Rico. However, basidiomycete allergens are not available for inclusion in allergy diagnostic panels. Therefore, we sought to confirm allergic sensitization to G. applanatum crude spore cytoplasmic extract through reactivity in serological assays and detection of immunoglobulin E (IgE)-binding polypeptides. Via an indirect ELISA, serological reactivity was compared between groups of individuals with different allergic profiles. Group 1 (n = 51) consisted of individuals with sIgE to the allergens included in the diagnostic panels; group 2 (n = 14) comprised individuals with no sIgE to the allergens tested; and group 3 (n = 22) included individuals with no allergic history. To visualize IgE-binding polypeptides, group 1 sera were examined via Western blotting (WB). Polypeptide bands with the highest reactivity were analyzed by mass spectrometry (MS) for putative identification. The serological reactivity of group 1 was significantly higher than that of group 3 in an indirect ELISA (p = 0.03). Sixty-five percent of group 1 individuals showed reactivity to polypeptide bands in WB. Bands of 81 and 56 kDa had the highest reactivity proportions among the reactive sera, followed by a 45-kDa band. MS analysis of these 3 polypeptides suggests that they are basidiomycete-derived enzymes with aconitate hydratase, catalase, and enolase functions. G. applanatum spores have allergenic components recognized by Puerto Rican individuals, which could eventually be considered as markers in cases of fungal allergy and be included in diagnostic allergen panels in Puerto Rico and tropical regions. © 2017 S. Karger AG, Basel.

Peptide Beacons: A New Design for Polypeptide-Based Optical Biosensors

PubMed Central

Oh, Kenneth J.; Cash, Kevin J.; Hugenberg, Verena; Plaxco, Kevin W.

2008-01-01

Phage display and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to their target. Using this effect to segregate a long-lived fluorophore from an electron transfer-based contact quencher, both covalently attached to the peptide, we have produced a robust optical sensor for anti-HIV antibodies. The binding-induced segregation of the fluorophore-quencher pair produces a six-fold increase in sensor emission, thus allowing us to readily detect as low as ∼250 pM of the target antibody. Because the sensor is based on binding-induced folding and a visible-light fluorophore, it is sufficiently selective to work directly in complex, contaminant-ridden samples such as saliva and blood. PMID:17461545

Heat shock of Escherichia coli increases binding of dnaK (the hsp70 homolog) to polypeptides by promoting its phosphorylation.

PubMed Central

Sherman, M Y; Goldberg, A L

1993-01-01

The "molecular chaperone", dnaK, is induced in Escherichia coli upon heat shock and promotes ATP-dependent refolding or degradation of damaged proteins. When cells were grown at 25 degrees C and disrupted, a small fraction of the dnaK bound to affinity columns containing unfolded polypeptides (e.g., a fusion protein named CRAG or casein) and could be dissociated by ATP-Mg2+. After shifting cells to 42 degrees C for 30 min, up to 5-fold more dnaK bound to these columns than after growth at 25 degrees C. This enhanced binding capacity was reversed after shifting cells back to 25 degrees C. It resulted from a covalent modification, which decreases dnaK's electrophoretic mobility and isoelectric point. This modification appears to be phosphorylation; after treatment with phosphatases, the ATP-eluted dnaK resembled the predominant form in electrophoretic and binding properties. In addition, after incubating cells with [32P]orthophosphate at 42 degrees C, the 32P-labeled dnaK bound quantitatively to the CRAG column, unlike the nonlabeled protein. Thus, the phosphorylated dnaK is a special form of the chaperone with enhanced affinity for unfolded proteins. Its accumulation at high temperatures may account for dnaK's function as the "cellular thermometer." Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8378342

Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases.

PubMed

Brewer, John M; Glover, Claiborne V C; Holland, Michael J; Lebioda, Lukasz

2003-05-01

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.

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.

Isolation and properties of the subunit form EF-1C of elongation factor 1 from Guerin epithelioma cells.

PubMed

Marcinkiewicz, C; Gałasiński, W

1993-01-01

EF-1C is a component of the aggregate EF-1B, consisting of the subunit forms EF-1A.EF-1C; it was isolated by dissociation of this aggregate in the presence of GTP. The subunit form EF-1C stimulates binding of aminoacyl-tRNA to ribosomes, catalysed by EF-1A, similarly as EF-1 beta gamma which stimulates the activity of EF-1 in other eukaryotic cells. EF-1C in the presence of 6 M urea was separated into two polypeptides. Polypeptide of molecular mass 32,000 Da is responsible for regeneration of the EF-1A.GTP active complex. Thermal sensitivity of EF-1A was much higher than that of EF-1B, thus a protective role of EF-1C in the EF-1A.EF-1C complex is suggested.

Uses of monoclonial antibody 8H9

DOEpatents

Cheung, Nai-Kong V.

2015-06-23

This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

Charybdotoxin is a new member of the K sup + channel toxin family that includes dendrotoxin I and mast cell degranulating peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schweitz, H.; Bidard, J.N.; Lazdunski, M.

1989-12-12

A polypeptide was identified in the venom of the scorpion Leiurus quinquestriatus hebraeus by its potency to inhibit the high affinity binding of the radiolabeled snake venom toxin dendrotoxin I ({sup 125}I-DTX{sub I}) to its receptor site. It has been purified, and its properties investigated by different techniques were found to be similar to those of MCD and DTX{sub I}, two polypeptide toxins active on a voltage-dependent K{sup +} channel. However, its amino acid sequence was determined, and it was shown that this toxin is in fact charybdotoxin (ChTX), a toxin classically used as a specific tool to block onemore » class of Ca{sup 2+}-activated K{sup +} channels. ChTX, DTX{sub I}, and MCD are potent convulsants and are highly toxic when injected intracerebroventricularly in mice. Their toxicities correlate well with their affinities for their receptors in rat brain. These three structurally different toxins release ({sup 3}H)GABA from preloaded synaptosomes, the efficiency order being DTX{sub I} > ChTX > MCD. Both binding and cross-linking experiments of ChTX to rat brain membranes and to the purified MCD/DTX{sub I} binding protein have shown that the {alpha}-subunit of the MCD/DTX{sub I}-sensitive K{sup +} channel protein also contains the ChTX binding sites. Binding sites for DTX{sub I}, MCD, and ChTX are in negative allosteric interaction. The results show that charybdotoxin belongs to the family of toxins which already includes the dendrotoxins and MCD, which are blockers of voltage-sensitive K{sup +} channels. ChTX is clearly not selective for Ca{sup 2+}-activated K{sup +} channel.« less

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.

Excimer-based peptide beacons: a convenient experimental approach for monitoring polypeptide-protein and polypeptide-oligonucleotide interactions.

PubMed

Oh, Kenneth J; Cash, Kevin J; Plaxco, Kevin W

2006-11-01

While protein-polypeptide and nucleic acid-polypeptide interactions are of significant experimental interest, quantitative methods for the characterization of such interactions are often cumbersome. Here we described a relatively simple means of optically monitoring such interactions using excimer-based peptide beacons (PBs). The design of PBs is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed with macromolecular targets. Using this binding-induced folding to segregate two pyrene moieties and therefore inhibit excimer formation, we have produced PBs directed against both anti-HIV antibodies and the retroviral transactive response (TAR) RNA hairpin. For both polypeptides, target recognition is accompanied by a roughly 2-fold decrease in excimer emission, thus allowing the detection of their respective targets at concentrations of a few nanomolar. Because excimer emission requires the formation of a tight, precisely oriented pyrene dimer, even relatively trivial binding-induced segregation reduces fluorescence significantly. This suggests that the PB approach will be suitable for monitoring a wide range of peptide-macromolecule recognition events. Moreover, the synthesis of excimer-based PBs utilizes commercially available modified pyrenes in a simple and well-established protocol, making the approach well suited for routine laboratory applications.

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4*

PubMed Central

Biswas-Fiss, Esther E.; Affet, Stephanie; Ha, Malissa; Biswas, Subhasis B.

2012-01-01

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. PMID:23144455

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

DOEpatents

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.

Chemically modified carbonic anhydrases useful in carbon capture systems

DOEpatents

Novick, Scott; Alvizo, Oscar

2013-01-15

The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

Chemically modified carbonic anhydrases useful in carbon capture systems

DOEpatents

Novick, Scott J; Alvizo, Oscar

2013-10-29

The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

Polypeptide having an amino acid replaced with N-benzylglycine

DOEpatents

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.

Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

NASA Technical Reports Server (NTRS)

Huang, J. F.; Teyton, L.; Harper, J. F.; Evans, M. L. (Principal Investigator)

1996-01-01

Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

Lignin blockers and uses thereof

DOEpatents

Yang, Bin [West Lebanon, NH; Wyman, Charles E [Norwich, VT

2011-01-25

Disclosed is a method for converting cellulose in a lignocellulosic biomass. The method provides for a lignin-blocking polypeptide and/or protein treatment of high lignin solids. The treatment enhances cellulase availability in cellulose conversion and allows for the determination of optimized pretreatment conditions. Additionally, ethanol yields from a Simultaneous Saccharification and Fermentation process are improved 5-25% by treatment with a lignin-blocking polypeptide and/or protein. Thus, a more efficient and economical method of processing lignin containing biomass materials utilizes a polypeptide/protein treatment step that effectively blocks lignin binding of cellulase.

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

Design and synthesis of type-III mimetics of ShK toxin

NASA Astrophysics Data System (ADS)

Baell, Jonathan B.; Harvey, Andrew J.; Norton, Raymond S.

2002-04-01

ShK toxin is a structurally defined, 35-residue polypeptide which blocks the voltage-gated Kv1.3 potassium channel in T-lymphocytes and has been identified as a possible immunosuppressant. Our interest lies in the rational design and synthesis of type-III mimetics of protein and polypeptide structure and function. ShK toxin is a challenging target for mimetic design as its binding epitope consists of relatively weakly binding residues, some of which are discontinuous. We discuss here our investigations into the design and synthesis of 1st generation, small molecule mimetics of ShK toxin and highlight any principles relevant to the generic design of type-III mimetics of continuous and discontinuous binding epitopes. We complement our approach with attempted pharmacophore-based database mining.

Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells.

PubMed

Rossi, Edmund A; Chang, Chien-Hsing; Losman, Michele J; Sharkey, Robert M; Karacay, Habibe; McBride, William; Cardillo, Thomas M; Hansen, Hans J; Qu, Zhengxing; Horak, Ivan D; Goldenberg, David M

2005-10-01

To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors. hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC. The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor-bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with (111)In. Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumor-to-nontumor ratios as early as 3 hours postinjection. The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.

An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure.

PubMed

Pfeiffer, M; Klein, A; Steinert, P; Schomburg, D

The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism.

Facilitating protein solubility by use of peptide extensions

DOEpatents

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

2013-09-17

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

Protein Arms in the Kinetochore-Microtubule Interface of the Yeast DASH Complex

PubMed Central

Miranda, JJ L.; King, David S.

2007-01-01

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface. PMID:17460120

Characterization and N-terminal sequencing of a calcium binding protein from the calcareous concretion organic matrix of the terrestrial crustacean Orchestia cavimana.

PubMed

Luquet, G; Testenière, O; Graf, F

1996-04-16

We extracted proteins from the organic matrix of calcareous concretions, which represents the calcium storage form in a terrestrial crustacean. Electrophoretic analyses of water-soluble organic-matrix proteinaceous components revealed 11 polypeptides, 6 of which are probably glycosylated. Among the unglycosylated proteins, we characterized a 23 kDa polypeptide, with an isoelectric point of 5.5, which is able to bind calcium. Its N-terminal sequence is rich in acidic amino acids (essentially aspartic acid). All these characteristics suggest its involvement in the calcium precipitation process within the successive layers of the organic matrix.

A split motor domain in a cytoplasmic dynein

PubMed Central

Straube, Anne; Enard, Wolfgang; Berner, Alexandra; Wedlich-Söldner, Roland; Kahmann, Regine; Steinberg, Gero

2001-01-01

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions. PMID:11566874

Bioresorbable polypeptide-based comb-polymers efficiently improves the stability and pharmacokinetics of proteins in vivo.

PubMed

Turabee, Md Hasan; Thambi, Thavasyappan; Lym, Jae Seung; Lee, Doo Sung

2017-03-28

Stimuli-responsive polypeptides are a promising class of biomaterials due to their tunable physicochemical and biological properties. Herein, a series of novel pH- and thermo-responsive block copolymers based on polypeptides were synthesized by ring-opening polymerization of γ-benzyl-l-glutamate-N-carboxyanhydride in the presence of poly(ethylene glycol)-diamine macroinitiator followed by aminolysis. The resulting polypeptide-based triblock copolymer, poly[(2-(dibutylamino)ethyl-l-glutamate)-co-(γ-benzyl-l-glutamate)]-poly(ethylene glycol)-b-poly[(2-(dibutylamino)ethyl-l-glutamate)-co-(γ-benzyl-l-glutamate)] (PNLG-co-PBLG-b-PEG-b-PBLG-co-PNLG), exists as a low viscous sol at low pH and temperature (≤pH 6.4, 25 °C) but it transforms to a soft gel under physiological conditions (pH 7.4 and 37 °C). The physical properties of the polypeptide gel can be tuned by controlling the ratio between hydrophobic PBLG and pH-sensitive PNLG blocks. The polypeptide-based copolymer did not show any noticeable cytotoxicity to fibroblast cells in vitro. It was found that subcutaneous injection of the polypeptide copolymer solution into the dorsal region of Sprague-Dawley (SD) rats formed a gel instantly without major inflammation. The gels were completely biodegraded in six weeks and found to be bioresorbable. Human growth hormone (hGH)-loaded polypeptide-based biodegradable copolymer sols readily formed a viscoelastic gel that inhibited an initial burst and prolonged the hGH release for one week. Overall, due to their bioresorbable and sustained release protein characteristics, polypeptide hydrogels may serve as viable platforms for therapeutic protein delivery and the surface tunable properties of polypeptide hydrogels can be exploited for other potential therapeutic proteins.

Investigating molecular crowding within nuclear pores using polarization-PALM

PubMed Central

Fu, Guo; Tu, Li-Chun; Zilman, Anton

2017-01-01

The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC’s internal organization consists of multiple dynamic environments with different local properties. PMID:28949296

Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

PubMed

Pawson, Tony; Kofler, Michael

2009-04-01

The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

Nucleotide sequence of the gene determining plasmid-mediated citrate utilization.

PubMed Central

Ishiguro, N; Sato, G

1985-01-01

The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2999087

Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

PubMed

Davis, J Q; Bennett, V

1993-01-01

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

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.

Evolving nucleotide binding surfaces

NASA Technical Reports Server (NTRS)

Kieber-Emmons, T.; Rein, R.

1981-01-01

An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA.

PubMed

Randall, Linda L; Henzl, Michael T

2010-06-01

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane-embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N-terminal residues 2 through 11 of SecA and the C-terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.

Compositions and Methods for the Treatment of Pierce's Disease

DOEpatents

Gupta, Goutam

2008-10-07

Chimeric anti-microbial proteins, compositions, and methods for the therapeutic and prophylactic treatment of plant diseases caused by the bacterial pathogen Xylella fastidiosa are provided. The anti-microbial proteins of the invention generally comprise a surface recognition domain polypeptide, capable of binding to a bacterial membrane component, fused to a bacterial lysis domain polypeptide, capable of affecting lysis or rupture of the bacterial membrane, typically via a fused polypeptide linker. In particular, methods and compositions for the treatment or prevention of Pierce's disease of grapevines are provided. Methods for the generation of transgenic Vitus vinefera plants expressing xylem-secreted anti-microbial chimeras are also provided.

Lignin-blocking treatment of biomass and uses thereof

DOEpatents

Yang, Bin [Hanover, NH; Wyman, Charles E [Norwich, VT

2009-10-20

Disclosed is a method for converting cellulose in a lignocellulosic biomass. The method provides for a lignin-blocking polypeptide and/or protein treatment of high lignin solids. The treatment enhances cellulase availability in cellulose conversion. Cellulase efficiencies are improved by the protein or polypeptide treatment. The treatment may be used in combination with steam explosion and acid prehydrolysis techniques. Hydrolysis yields from lignin containing biomass are enhanced 5-20%, and enzyme utilization is increased from 10% to 50%. Thus, a more efficient and economical method of processing lignin containing biomass materials utilizes a polypeptide/protein treatment step that effectively blocks lignin binding of cellulase.

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

PubMed

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

2015-04-14

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway

PubMed Central

Sukumaran, Salil K.; Blau-Wasser, Rosemarie; Rohlfs, Meino; Gallinger, Christoph; Schleicher, Michael; Noegel, Angelika A

2015-01-01

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction. PMID:25607232

Impact of membrane curvature on amyloid aggregation.

PubMed

Terakawa, Mayu S; Lin, Yuxi; Kinoshita, Misaki; Kanemura, Shingo; Itoh, Dai; Sugiki, Toshihiko; Okumura, Masaki; Ramamoorthy, Ayyalusamy; Lee, Young-Ho

2018-04-28

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs. We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

PubMed

Mezö, G; Hudecz, F; Kajtár, J; Szókán, G; Szekerke, M

1989-10-01

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n

Purification and characterization of human pancreatic polypeptide expressed in E. coli.

PubMed

Griko, Y V; Kapanadze, M D

1995-08-04

The region of cDNA encoding human pancreatic polypeptide (hPP) was obtained by polymerase chain reaction (PCR) and subcloned into an expression vector. The pancreatic polypeptide gene was expressed in Escherichia coli in two versions: as a cleavable fusion protein with IgG-binding synthetic ZZ domains of protein A from Staphylococcus aureus or with the 1-48 fragment of lambda Cro repressor. Site-specific hydrolysis by hydroxylamine was used to cleave the fusion protein, releasing the human polypeptide. The structure of the obtained hPP has been studied by scanning microcalorimetry and circular dichroism spectrometry. It has been shown that hPP in solutions close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is stable at 20 degrees C and co-operatively breaks down upon heating from this temperature.

Role of Side-Chain Molecular Features in Tuning Lower Critical Solution Temperatures (LCSTs) of Oligoethylene Glycol Modified Polypeptides.

PubMed

Gharakhanian, Eric G; Deming, Timothy J

2016-07-07

A series of thermoresponsive polypeptides has been synthesized using a methodology that allowed facile adjustment of side-chain functional groups. The lower critical solution temperature (LCST) properties of these polymers in water were then evaluated relative to systematic molecular modifications in their side-chains. It was found that in addition to the number of ethylene glycol repeats in the side-chains, terminal and linker groups also have substantial and predictable effects on cloud point temperatures (Tcp). In particular, we found that the structure of these polypeptides allowed for inclusion of polar hydroxyl groups, which significantly increased their hydrophilicity and decreased the need to use long oligoethylene glycol repeats to obtain LCSTs. The thioether linkages in these polypeptides were found to provide an additional structural feature for reversible switching of both polypeptide conformation and thermoresponsive properties.

Biopolymers Containing Unnatural Amino Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty aminomore » acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.« less

Biopolymers Containing Unnatural Building Blocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter G.

2013-06-30

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty aminomore » acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.« less

NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.

PubMed Central

Neuhauser, W; Haltrich, D; Kulbe, K D; Nidetzky, B

1997-01-01

During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase. PMID:9307017

Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors.

PubMed Central

Guidotti, A; Forchetti, C M; Corda, M G; Konkel, D; Bennett, C D; Costa, E

1983-01-01

A brain polypeptide termed diazepam-binding inhibitor (DBI) and thought to be chemically and functionally related to the endogenous effector of the benzodiazepine recognition site was purified to homogeneity. This peptide gives a single band of protein on NaDodSO4 and acidic urea gel electrophoresis. A single UV-absorbing peak was obtained by HPLC using three different columns and solvent systems. DBI has a molecular mass of approximately equal to 11,000 daltons. Carboxyl-terminus analysis shows that tyrosine is the only residue while the amino-terminus was blocked. Cyanogen bromide treatment of DBI yields three polypeptide fragments, and the sequences of two of them have been determined for a total of 45 amino acids. DBI is a competitive inhibitor for the binding of [3H]diazepam, [3H]flunitrazepam, beta-[3H]carboline propyl esters, and 3H-labeled Ro 15-1788. The Ki for [3H]-diazepam and beta-[3H]carboline binding were 4 and 1 microM, respectively. Doses of DBI that inhibited [3H]diazepam binding by greater than 50% fail to change [3H]etorphine, gamma-amino[3H]butyric acid, [3H]-quinuclidinyl benzilate, [3H]dihydroalprenolol, [3H]adenosine, and [3H]imipramine binding tested at their respective Kd values. DBI injected intraventricularly at doses of 5-10 nmol completely reversed the anticonflict action of diazepam on unpunished drinking and, similar to the anxiety-inducing beta-carboline derivative FG 7142 (beta-carboline-3-carboxylic acid methyl ester), facilitated the shock-induced suppression of drinking by lowering the threshold for this response. Images PMID:6304714

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

PubMed

Kylväjä, Riikka; Ojalehto, Tuomas; Kainulainen, Veera; Virkola, Ritva; Westerlund-Wikström, Benita

2016-08-04

Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

Investigation of light-induced conformation changes in spiropyran-modified succinylated poly(L-lysine).

PubMed

Cooper, T M; Stone, M O; Natarajan, L V; Crane, R L

1995-08-01

To determine the maximum range of coupling between side-chain photochromism and polypeptide conformation change, we modified the carboxylate side chains of succinylated poly(L-lysine) with a spiropyran to form polypeptide I. The extent of modification was determined to be 35.5%. The spacer group length between the polypeptide alpha-carbon and the dye was 12 atoms, providing minimum polypeptide-dye interaction. Conformation changes were monitored by circular dichroism as a function of light adaptation and solvent composition (hexafluoroisopropanol [HFIP] vs trifluoroethanol [TFE]). Under all solvent compositions, the dark-adapted dye was in the merocyanine form. Light adaptation by visible light converted the dye to the spiropyran form. When dissolved in TFE, I adopted a helical conformation insensitive to light adaptation. With increasing percentage HFIP, a solvent-induced helix-to-coil transition was observed around 80% (vol/vol) HFIP. At 100% HFIP, both light- and dark-adapted forms of I were in the coil state. Near the midpoint of the solvent-induced helix-to-coil transition, light adaptation caused conformation changes. Applying helix-to-coil transition theory, we measured a statistically significant difference in coil segment-HFIP binding constant for light- vs dark-adapted solutions (6.38 +/- 0.03 M-1 vs 6.56 +/- 0.03 M-1), but not for the nucleation parameter sigma (1.2 +/- 0.4 10(-3) vs 1.3 +/- 0.3 x 10(-3). The small binding constant difference translated to a light-induced binding energy difference of 17 cal/mol/monomer. Near the midpoint of the helix-to-coil transition, collective interactions between monomer units made possible the translation of a small energy difference (less than RT) into large macromolecular conformation changes.(ABSTRACT TRUNCATED AT 250 WORDS)

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

PubMed Central

Lundin, Victor F.; Stirling, Peter C.; Gomez-Reino, Juan; Mwenifumbo, Jill C.; Obst, Jennifer M.; Valpuesta, José M.; Leroux, Michel R.

2004-01-01

Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides. PMID:15070724

Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

DOEpatents

Tsien, Roger Y [La Jolla, CA; Wang, Lei [San Diego, CA

2008-07-01

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.

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.

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

PubMed Central

Cooper, J A; Kashishian, A

1993-01-01

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

CIP1 polypeptides and their uses

DOEpatents

Foreman, Pamela [Los Altos, CA; Van Solingen, Pieter [Naaldwijk, NL; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA

2011-04-12

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Using porphyrin-amino acid pairs to model the electrochemistry of heme proteins: experimental and theoretical investigations.

PubMed

Samajdar, Rudra N; Manogaran, Dhivya; Yashonath, S; Bhattacharyya, Aninda J

2018-04-18

Quasi reversibility in electrochemical cycling between different oxidation states of iron is an often seen characteristic of iron containing heme proteins that bind dioxygen. Surprisingly, the system becomes fully reversible in the bare iron-porphyrin complex: hemin. This leads to the speculation that the polypeptide bulk (globin) around the iron-porphyrin active site in these heme proteins is probably responsible for the electrochemical quasi reversibility. To understand the effect of such polypeptide bulk on iron-porphyrin, we study the interaction of specific amino acids with the hemin center in solution. We choose three representative amino acids-histidine (a well-known iron coordinator in bio-inorganic systems), tryptophan (a well-known fluoroprobe for proteins), and cysteine (a redox-active organic molecule). The interactions of these amino acids with hemin are studied using electrochemistry, spectroscopy, and density functional theory. The results indicate that among these three, the interaction of histidine with the iron center is strongest. Further, histidine maintains the electrochemical reversibility of iron. On the other hand, tryptophan and cysteine interact weakly with the iron center but disturb the electrochemical reversibility by contributing their own redox active processes to the system. Put together, this study attempts to understand the molecular interactions that can control electrochemical reversibility in heme proteins. The results obtained here from the three representative amino acids can be scaled up to build a heme-amino acid interaction database that may predict the electrochemical properties of any protein with a defined polypeptide sequence.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

A further insight into the biosorption mechanism of Au(III) by infrared spectrometry

PubMed Central

2011-01-01

Background The interactions of microbes with metal ions form an important basis for our study of biotechnological applications. Despite the recent progress in studying some properties of Au(III) adsorption and reduction by Bacillus megatherium D01 biomass, there is still a need for additional data on the molecular mechanisms of biosorbents responsible for their interactions with Au(III) to have a further insight and to make a better exposition. Results The biosorption mechanism of Au(III) onto the resting cell of Bacillus megatherium D01 biomass on a molecular level has been further studied here. The infrared (IR) spectroscopy on D01 biomass and that binding Au(III) demonstrates that the molecular recognition of and binding to Au(III) appear to occur mostly with oxygenous- and nitrogenous-active groups of polysaccharides and proteins in cell wall biopolymers, such as hydroxyl of saccharides, carboxylate anion of amino-acid residues (side-chains of polypeptide backbone), peptide bond (amide I and amide II bands), etc.; and that the active groups must serve as nucleation sites for Au(0) nuclei growth. A further investigation on the interactions of each of the soluble hydrolysates of D01, Bacillus licheniformis R08, Lactobacillus sp. strain A09 and waste Saccharomyces cerevisiae biomasses with Au(III) by IR spectrometry clearly reveals an essential biomacromolecule-characteristic that seems the binding of Au(III) to the oxygen of the peptide bond has caused a significant, molecular conformation-rearrangement in polypeptide backbones from β-pleated sheet to α-helices and/or β-turns of protein secondary structure; and that this changing appears to be accompanied by the occurrence, in the peptide bond, of much unbound -C=O and H-N- groups, being freed from the inter-molecular hydrogen-bonding of the β-pleated sheet and carried on the helical forms, as well as by the alternation in side chain steric positions of protein primary structure. This might be reasonably expected to result in higher-affinity interactions of peptide bond and side chains with Au(III). Conclusions The evidence suggests that the polypeptides appear to be activated by the intervention of Au(III) via the molecular reconformation and in turn react upon Au(III) actively and exert profound impacts on the course of Au(0) nucleation and crystal growth. PMID:22032692

Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains.

PubMed

Yang, S W; Becker, F F; Chan, J Y

1990-10-25

DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel electrophoresis of their AMP-binding domains in the trypsin-digested protein fragments. The results also suggested that these isozymes have been derived from the same primordial DNA sequence or from the same precursor protein. The purification scheme and the data obtained will be instrumental for the further elucidation of the biological roles of various DNA ligases from human cells.

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.

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.

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.

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.

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.

Exploration of the immunoreactivity of the Traditional Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide

PubMed Central

Gu, Yu-Chun; Chen, De-Zhen

1997-01-01

AIM: To study the immunoreactivity of the Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide (VIP) and its therapeutic mechanism. METHODS: The immunoreactivity of the Chinese medicine Shenrouyangzhentang to VIP was detected in the plasma of 20 normal people and 20 patients with Piyinxu (Spleen Yin deficiency) using the radioimmunoassay (RIA) method. RESULTS: The maximum binding rate B0/T was 53.29%, the non-specific binding rate N0/T was 1.170%, and the VIP standard curve was Y = 0.81983 + 0.44319X - 0.28927X2, R2 = 0.990. The VIP content in Shenrouyangzhentang was 106.6 ng/L ± 20 ng/L), while it was 90.16 ng/L ± 15 ng/L in normal human plasma and 63.25 ng/L ± 11 ng/L in the plasma of Pixinxu patients. The difference between normal plasma and Pixinxu patient plasma was statistically significant (P < 0.05). CONCLUSION: The Chinese medicine Shenrouyangzhentang demonstrated VIP immunoreactivity similar to that of normal plasma. The (vasoactive intestinal polypeptide) VIP content in Pixinxu patient plasma was lower than that in healthy subjects (P < 0.05). PMID:27041949

Inhibition and Ultraviolet-Induced Chemical Modification of UDP-Glucose:(1,3)-β-Glucan (Callose) Synthase by Chlorpromazine 1

PubMed Central

Harriman, Robert W.; Shao, Ai-Ping; Wasserman, Bruce P.

1992-01-01

UDP-glucose:(1,3)-β-glucan (callose) synthase (CS) from storage tissue of red beet (Beta vulgaris L.) was strongly inhibited by the phenothiazine drug chlorpromazine (CPZ). In the absence of ultraviolet irradiation, CPZ was a noncompetitive inhibitor with 50% inhibitory concentration values for plasma membrane and solubilized CS of 100 and 90 μm, respectively. Both the Ca2+- and Mg2+- stimulated components of CS activity were affected. CPZ inhibition was partially alleviated at saturating levels of Ca2+, but not Mg2+, suggesting that CPZ interferes with the Ca2+-binding site of CS. Binding experiments with [14C]CPZ, however, showed strong non-specific partitioning of CPZ into the plasma membrane, providing evidence that perturbation of the membrane environment is probably the predominant mode of inhibition. Ultraviolet irradiation at 254 nm markedly enhanced CPZ inhibition, with complete activity loss following exposure to 4 μm CPZ for 2 min. Inhibition followed a pseudo-first order mechanism with at least three CPZ binding sites per CS complex. Under these conditions, [3H]CPZ was covalently incorporated into plasma membrane preparations by a free radical mechanism; however, polypeptide labeling profiles showed labeling to be largely nonspecific, with many polypeptides labeled even at [3H]CPZ levels as low as 1 μm, and with boiled membranes. Although CPZ is one of the most potent known inhibitors of CS, its use as a photolabel will require a homogeneous CS complex or establishment of conditions that protect against the interaction of CPZ with specific binding sites located on various polypeptide components of the CS complex. PMID:16653219

Probing binding hot spots at protein-RNA recognition sites.

PubMed

Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

2016-01-29

We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

PubMed Central

Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

1989-01-01

Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

Protein scaffolds for selective enrichment of metal ions

DOEpatents

He, Chuan; Zhou, Lu; Bosscher, Michael

2016-02-09

Polypeptides comprising high affinity for the uranyl ion are provided. Methods for binding uranyl using such proteins are likewise provided and can be used, for example, in methods for uranium purification or removal.

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles.

PubMed

Lerch, Mirjam; Gafner, Verena; Bader, Reto; Christen, Barbara; Folkers, Gerd; Zerbe, Oliver

2002-10-04

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34. The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.

Adjunct therapy for type 1 diabetes mellitus.

PubMed

Lebovitz, Harold E

2010-06-01

Insulin replacement therapy in type 1 diabetes mellitus (T1DM) is nonphysiologic. Hyperinsulinemia is generated in the periphery to achieve normal insulin concentrations in the liver. This mismatch results in increased hypoglycemia, increased food intake with weight gain, and insufficient regulation of postprandial glucose excursions. Islet amyloid polypeptide is a hormone synthesized in pancreatic beta cells and cosecreted with insulin. Circulating islet amyloid polypeptide binds to receptors located in the hindbrain and increases satiety, delays gastric emptying and suppresses glucagon secretion. Thus, islet amyloid polypeptide complements the effects of insulin. T1DM is a state of both islet amyloid polypeptide and insulin deficiency. Pramlintide, a synthetic analog of islet amyloid polypeptide, can replace this hormone in patients with T1DM. When administered as adjunctive therapy to such patients treated with insulin, pramlintide decreases food intake and causes weight loss. Pramlintide therapy is also associated with suppression of glucagon secretion and delayed gastric emptying, both of which decrease postprandial plasma glucose excursions. Pramlintide therapy improves glycemic control and lessens weight gain. Agents that decrease intestinal carbohydrate digestion (alpha-glucosidase inhibitors) or decrease insulin resistance (metformin) might be alternative adjunctive therapies in T1DM, though its benefits are marginally supported by clinical data.

Biomimetic assembly of polypeptide-stabilized CaCO(3) nanoparticles.

PubMed

Zhang, Zhongping; Gao, Daming; Zhao, Hui; Xie, Chenggen; Guan, Guijian; Wang, Dapeng; Yu, Shu-Hong

2006-05-04

In this paper, we report a simple polypeptide-directed strategy for fabricating large spherical assembly of CaCO(3) nanoparticles. Stepwise growth and assembly of a large number of nanoparticles have been observed, from the formation of an amorphous liquidlike CaCO(3)-polypeptide precursor, to the crystallization and stabilization of polypeptide-capped nanoparticles, and eventually, the spherical assembly of nanoparticles. The "soft" poly(aspartate)-capping layer binding on a nanoparticle surface resulted in the unusual soft nature of nanoparticle assembly, providing a reservoir of primary nanoparticles with a moderate mobility, which is the basis of a new strategy for reconstructing nanoparticle assembly into complex nanoparticle architectures. Moreover, the findings of the secondary assembly of nanoparticle microspheres and the morphology transformation of nanoparticle assembly demonstrate a flexible and controllable pathway for manipulating the shapes and structures of nanoparticle assembly. In addition, the combination of the polypeptide with a double hydrophilic block copolymer (DHBC) allows it to possibly further control the shape and complexity of the nanoparticle assembly. A clear perspective is shown here that more complex nanoparticle materials could be created by using "soft" biological proteins or peptides as a mediating template at the organic-inorganic interface.

Role of Monomer Sequence, Hydrogen Bonding and Mesoscale Architecture in Marine Antifouling Coatings

NASA Astrophysics Data System (ADS)

Segalman, Rachel

Polypeptoids are non-natural, sequence specific polymers that offer the opportunity to probe the effect of monomer sequence, chirality, and chain shape on self-assembly and surface properties. Additionally, polypeptoid synthesis is more scaleable than traditional polypeptides suggesting their utility in large area applications. We have designed efficient marine anti-fouling coatings by using triblock copolymer scaffolds to which polypeptoids are tethered in order to tune both the modulus and surface energies with great precision. Surprisingly, when short sequences are tethered to a polymer backbone, polypeptoids consistently outperform analogous polypeptides in antifouling properties. We hypothesize that the hydrogen bonding inherent to the polypeptide backbone drives the observed differences in performance. We also find that the polymer scaffold housing the polypeptoids also plays a crucial role in directing surface presentation and therefore the overall coating properties.

Tunable drug loading and release from polypeptide multilayer nanofilms

PubMed Central

Jiang, Bingbing; Li, Bingyun

2009-01-01

Polypeptide multilayer nanofilms were prepared using electrostatic layer-by-layer self-assembly nanotechnology. Small charged drug molecules (eg, cefazolin, gentamicin, and methylene blue) were loaded in polypeptide multilayer nanofilms. Their loading and release were found to be pH-dependent and could also be controlled by changing the number of film layers and drug incubation time, and applying heat-treatment after film formation. Antibioticloaded polypeptide multilayer nanofilms showed controllable antibacterial properties against Staphylococcus aureus. The developed biodegradable polypeptide multilayer nanofilms are capable of loading both positively- and negatively-charged drug molecules and promise to serve as drug delivery systems on biomedical devices for preventing biomedical device-associated infection, which is a significant clinical complication for both civilian and military patients. PMID:19421369

Chirality-selected phase behaviour in ionic polypeptide complexes

DOE PAGES

Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; ...

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

Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

PubMed

de Souza, Tatiana de Arruda Campos Brasil; Graça-de Souza, Viviane Krominski; Lancheros, César Armando Contreras; Monteiro-Góes, Viviane; Krieger, Marco Aurélio; Goldenberg, Samuel; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2011-03-01

In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.

Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing.

PubMed

Wang, Ziqiang; Liu, Qing; Lu, Jinhua; Fan, Ping; Xie, Weidong; Qiu, Wei; Wang, Fan; Hu, Guangnan; Zhang, Yaou

2016-12-16

Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones

PubMed Central

Keefer, Kathryn M.; True, Heather L.

2016-01-01

The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders. PMID:27828954

Polypeptides and polyaminoacids in drug delivery.

PubMed

González-Aramundiz, José Vicente; Lozano, María Victoria; Sousa-Herves, Ana; Fernandez-Megia, Eduardo; Csaba, Noemi

2012-02-01

Advances achieved over the last few years in drug delivery have provided novel and versatile possibilities for the treatment of various diseases. Among the biomaterials applied in this field, it is worth highlighting the increasing importance of polyaminoacids and polypeptides. The appealing properties of these polymers are very promising for the design of novel compositions in a variety of drug delivery applications. This review provides an overview on the general characteristics of polyaminoacids and polypeptides and briefly discusses different synthetic pathways for their production. This is followed by a detailed description of different drug delivery applications of these polymers, emphasizing those examples that already reached advanced preclinical development or have entered clinical trials. Polyaminoacids and polypeptides are gaining much attention in drug delivery due to their exceptional properties. Their application as polymers for drug delivery purposes has been sped up by the significant achievements related to their synthesis. Certainly, cancer therapy has benefited the most from these advances, although other fields such as vaccine delivery and alternative administration routes are also being successfully explored. The design of new entities based on polyaminoacids and polypeptides and the improved insight gained in drug delivery guarantee exciting findings in the near future.

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

PubMed Central

Gil-Moreno, Selene; Jiménez-Martí, Elena; Palacios, Òscar; Zerbe, Oliver; Dallinger, Reinhard; Capdevila, Mercè; Atrian, Sílvia

2015-01-01

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion. PMID:26703589

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

PubMed Central

Al-Khouri, Anna Maria; Paule, Marvin R.

2002-01-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

PubMed

Al-Khouri, Anna Maria; Paule, Marvin R

2002-02-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

Activated protein kinase C binds to intracellular receptors in rat hepatocytes.

PubMed

Robles-Flores, M; García-Sáinz, J A

1993-12-01

The aim of this study was to identify in rat hepatocytes cellular polypeptides that bind protein kinase C (PKC) and may influence its activity and its compartmentation. At least seven proteins, with apparent M(r) values between 12,000 and 36,000, that behave like Receptors for Activated C-Kinase (RACKs) were found in the Triton-X-100-insoluble fraction of these cells; i.e. PKC bound to these polypeptides when it was in its active form. RACKS seem to be PKC substrates. Studies using isotype-specific PKC antibodies suggested some selectivity of RACKs, i.e. RACKs in the M(r) approximately 28,000-36,000 region bound PKC-alpha and PKC-beta in the presence of phosphatidylserine, diolein and Ca2+, whereas those of M(r) approximately 12,000-14,000 bound all isoforms studied, and, in contrast with the other RACKs, they did this even in the absence of Ca2+. Peptide I (KGDYEKILVALCGGN), which has a sequence suggested to be involved in the PKC-RACKs interaction [Mochly-Rosen, Khaner, Lopez and Smith (1991) J. Biol. Chem. 266, 14866-14868], inhibited PKC activity. Preincubation of RACKs with antisera directed against peptide I prevented PKC binding to them. The data suggest that peptide I blocks PKC binding to RACKs by two mechanisms: inhibition of PKC activity and competition with a putative binding site.

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

PubMed Central

Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M.; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet, Jaume; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva, Baldo; Sanz, Laura; Blanco, Francisco J.; Alvarez-Vallina, Luis

2016-01-01

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas. PMID:27345490

Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.

PubMed Central

Bischoff, F R; Krebber, H; Smirnova, E; Dong, W; Ponstingl, H

1995-01-01

RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins. Images PMID:7882974

Monosaccharide transporter of the human erythrocyte. Characterization of an improved preparation.

PubMed

Baldwin, S A; Baldwin, J M; Lienhard, G E

1982-08-03

The human erythrocyte monosaccharide transporter has been purified through the use of the dialyzable detergent octyl glucoside. It was found that the transporter denatures in the detergent and that the rate of this process could be reduced by increasing the ratio of phospholipid to detergent. The transporter was obtained in higher yield and with a higher specific activity for cytochalasin B binding than has been previously reported. Scatchard plot analysis of cytochalasin B binding to the reconstituted preparations gave a dissociation constant of 1.5 X 10(-7) M, and there were found to be 15.3 nmol of sites/mg of protein. On the basis of a value of 46 000 for the molecular weight of the polypeptide, this specific activity corresponds to 0.70 site/polypeptide chain; and there are reasons to believe that the value of the stoichiometry may be one site per functional transporter polypeptide. The complete amino acid composition and the N- and C-terminal residues of the transporter have been determined. Both the intact transporter and transporter that had been partially depleted of carbohydrate by treatment with endo-beta-galactosidase were found to migrate anomalously upon sodium dodecyl sulfate--polyacrylamide gel electrophoresis, relative to the behavior of standard proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kahoun, J.R.; Ruoho, A.E.

A carrier-free radioiodinated cocaine photoaffinity label, (-)-3-({sup 125}I)iodo-4-azidococaine (({sup 125}I)IACoc), has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM ({sup 125}I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. ({sup 125}I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 {mu}M imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the ({sup 125}I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigmamore » photolabel azido-({sup 3}H)DTG. Kinetic analysis of ({sup 125}I)IACoc binding to rat liver microsomes revealed two sites with K{sub d} values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, ({sup 125}I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.« less

New molecular features of cowpea bean (Vigna unguiculata, l. Walp) β-vignin.

PubMed

de Souza Ferreira, Ederlan; Capraro, Jessica; Sessa, Fabio; Magni, Chiara; Demonte, Aureluce; Consonni, Alessandro; Augusto Neves, Valdir; Maffud Cilli, Eduardo; Duranti, Marcello; Scarafoni, Alessio

2018-02-01

Cowpea seed β-vignin, a vicilin-like globulin, proved to exert various health favourable effects, including blood cholesterol reduction in animal models. The need of a simple scalable enrichment procedure for further studies for tailored applications of this seed protein is crucial. A chromatography-independent fractionation method allowing to obtain a protein preparation with a high degree of homogeneity was used. Further purification was pursued to deep the molecular characterisation of β-vignin. The results showed: (i) differing glycosylation patterns of the two constituent polypeptides, in agreement with amino acid sequence features; (ii) the seed accumulation of a gene product never identified before; (iii) metal binding capacity of native protein, a property observed only in few other legume seed vicilins.

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

PubMed

Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

PubMed Central

Sun, Weina; McCrory, Thomas S.; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell

2014-01-01

ABSTRACT Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. PMID:25210190

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

PubMed Central

Hwang, Byung Joon; Toering, Stephanie; Francke, Uta; Chu, Gilbert

1998-01-01

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin. PMID:9632823

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, R.; Wilton, R.; Cuff, M. E.

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes butmore » have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.« less

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.

Molecular description of the LCST behavior of an elastin-like polypeptide.

PubMed

Li, Nan K; García Quiroz, Felipe; Hall, Carol K; Chilkoti, Ashutosh; Yingling, Yaroslava G

2014-10-13

Elastin-like polypeptides (ELPs) with the repeat sequence of VPGVG are widely used as a model system for investigation of lower critical solution temperature (LCST) transition behavior. In this paper, the effect of temperature on the structure, dynamics and association of (VPGVG)18 in aqueous solution is investigated using atomistic molecular dynamics simulations. Our simulations show that as the temperature increases the ELP backbones undergo gradual conformational changes, which are attributed to the formation of more ordered secondary structures such as β-strands. In addition, increasing temperature changes the hydrophobicity of the ELP by exposure of hydrophobic valine-side chains to the solvent and hiding of proline residues. Based on our simulations, we conclude that the transition behavior of (VPGVG)18 can be attributed to a combination of thermal disruption of the water network that surrounds the polypeptide, reduction of solvent accessible surface area of the polypeptide, and increase in its hydrophobicity. Simulations of the association of two (VPGVG)18 molecules demonstrated that the observed gradual changes in the structural properties of the single polypeptide chain are enough to cause the aggregation of polypeptides above the LCST. These results lead us to propose that the LCST phase behavior of poly(VPGVG) is a collective phenomenon that originates from the correlated gradual changes in single polypeptide structure and the abrupt change in properties of hydration water around the peptide and is a result of a competition between peptide-peptide and peptide-water interactions. This is a computational study of an important intrinsically disordered peptide system that provides an atomic-level description of structural features and interactions that are relevant in the LCST phase behavior.

Brachytherapy Using Elastin-Like Polypeptides with (131)I Inhibit Tumor Growth in Rabbits with VX2 Liver Tumor.

PubMed

Liu, Xinpei; Shen, Yiming; Zhang, Xuqian; Lin, Rui; Jia, Qiang; Chang, Yixiang; Liu, Wenge; Liu, Wentian

2016-10-01

Brachytherapy is a targeted type of radiotherapy utilized in the treatment of cancers. Elastin-like polypeptides are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy. To explore the feasibility and application of brachytherapy for VX2 liver tumor using elastin-like polypeptides with (131)I so as to provide reliable experimental evidence for a new promising treatment of liver cancer. Elastin-like polypeptide as carrier was labeled with (131)I using the iodogen method. Ten eligible rabbits with VX2 liver tumor were randomly divided into the treatment group (n = 5) and control group (n = 5). The treatment group received brachytherapy using elastin-like polypeptide with (131)I, and in the control group, elastin-like polypeptide was injected into the VX2 liver tumor as a control. Periodic biochemical and imaging surveillances were required to assess treatment efficacy. The stability of elastin-like polypeptide with (131)I in vitro was maintained at over 96.8 % for 96 h. Biochemistry and imaging indicated brachytherapy using elastin-like polypeptide with (131)I for liver tumor can improve liver function and inhibit tumor growth (P < 0.05). Elastin-like polypeptide can be an ideal carrier of (131)I and have high labeling efficiency, radiochemical purity and stability. Brachytherapy using elastin-like polypeptide with (131)I for liver tumor is a useful therapy that possesses high antitumor efficacy advantages.

Expression of eukaryotic polypeptides in chloroplasts

DOEpatents

Mayfield, Stephen P.

2013-06-04

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

Structure-based receptor MIMICS targeted against bacterial superantigen toxins

DOEpatents

Gupta, Goutam [Santa Fe, NM; Hong-Geller, Elizabeth [Los Alamos, NM; Shiflett, Patrick R [Los Alamos, NM; Lehnert, Nancy M [Albuquerque, NM

2009-08-18

The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides.

PubMed

Jean, D H; Albers, R W; Koval, G J

1975-02-10

Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase.

Lipid functions in cytochrome bc complexes: an odd evolutionary transition in a membrane protein structure

PubMed Central

Hasan, S. Saif; Cramer, William A.

2012-01-01

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b6f and the yeast bc1 complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b6f complex overlap four sites in the Chlamydomonas reinhardtii algal b6f complex and four in the yeast bc1 complex. The proposed lipid functions include: (i) interfacial–interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron–sulphur protein (ISP), and four small subunits in the boundary ‘picket fence’); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a ‘latch’ to photosystem I provided by the β-carotene chain protruding through the ‘picket fence’; (v) presence of a lipid and chlorophyll a chlorin ring in b6f in place of the eighth helix in the bc1 cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b6f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization. PMID:23148267

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells.

PubMed

Gitman, A G; Kahane, I; Loyter, A

1985-05-21

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.

Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

PubMed

Renault, L

2016-01-01

β-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/β-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling. © 2016 Elsevier Inc. All rights reserved.

Prosegment of tripeptidyl peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.

PubMed

Golabek, Adam A; Dolzhanskaya, Natalia; Walus, Marius; Wisniewski, Krystyna E; Kida, Elizabeth

2008-06-13

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme*

PubMed Central

Golabek, Adam A.; Dolzhanskaya, Natalia; Walus, Marius; Wisniewski, Krystyna E.; Kida, Elizabeth

2008-01-01

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment. PMID:18411270

Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes.

PubMed

Castillo, Virginia; Graña-Montes, Ricardo; Sabate, Raimon; Ventura, Salvador

2011-06-01

In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter.

PubMed Central

Briegel, K; Hentsch, B; Pfeuffer, I; Serfling, E

1991-01-01

The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a 'perfect' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and, as a functional consequence, to the activity of the 'converted' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene. Images PMID:1945879

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros.

PubMed

Hemmi, Hikaru; Ishibashi, Jun; Tomie, Tetsuya; Yamakawa, Minoru

2003-06-20

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain.

PubMed

Wu, R; Wilton, R; Cuff, M E; Endres, M; Babnigg, G; Edirisinghe, J N; Henry, C S; Joachimiak, A; Schiffer, M; Pokkuluri, P R

2017-04-01

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport. © 2017 The Protein Society.

The role of aromatic phenylalanine residues in binding carotenoid to light-harvesting model and wild-type complexes.

PubMed

García-Martín, A; Pazur, A; Wilhelm, B; Silber, M; Robert, B; Braun, P

2008-09-26

The mode of carotenoid (Crt) binding to polypeptide and specifying its function is as yet largely unknown. Statistical analysis of major photosystems I and II suggests that aromatic residues make up a significant part of the Crt binding pockets. Phenylalanine residues ensure approximately 25%--at some carbon atoms even up to 40%--of the total contacts with Crts. By use of an alanine-leucine model transmembrane helix that replaces the native helix of the bacterial light-harvesting complex 2 (LH2) alpha-subunit, we study the effects of polypeptide residues on cofactor binding in a model sequence context. Here, it is shown that phenylalanine residues located in the close vicinity of the Crts' polyene backbone significantly contribute to the binding of the Crt to the model protein. The replacement of a phenylalanine with leucine in the model helix results in significant reduction in the complexes' Crt content. This effect is strongly enhanced by the removal of a second phenylalanine in close vicinity to the Crt, i.e., of the wild-type (WT) beta-subunit. Remarkably, the mutation of only two phenylalanine residues in the LH2 WT sequence, alpha-Phe at position -12 and beta-Phe at -8, results in the loss of nearly 50% of functional Crt. Resonance Raman spectra indicate that the Crt conformation is fundamentally altered by the absence of the phenylalanines' aromatic side chains, suggesting that they lock the Crt into a precise, well-defined configuration. Thus, binding and specific functionalisation of Crt in the model and WT light-harvesting complex is reliant on the aromatic residue phenylalanine. The use of the light-harvesting complex as a model system thus substantiates the notion that the aromatic residue phenylalanine is a key factor for the binding of Crt to transmembrane proteins.

Highly stable beta-class carbonic anhydrases useful in carbon capture systems

DOEpatents

Alvizo, Oscar; Benoit, Mike; Novick, Scott

2013-04-16

The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

Highly stable beta-class carbonic anhydrases useful in carbon capture systems

DOEpatents

Alvizo, Oscar; Benoit, Michael R; Novick, Scott J

2013-08-20

The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

PubMed Central

Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.

2016-01-01

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility. PMID:27222581

A Plant Small Polypeptide Is a Novel Component of DNA-Binding Protein Phosphatase 1-Mediated Resistance to Plum pox virus in Arabidopsis1[C][W

PubMed Central

Castelló, María José; Carrasco, Jose Luis; Navarrete-Gómez, Marisa; Daniel, Jacques; Granot, David; Vera, Pablo

2011-01-01

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis. PMID:22021419

Bovine seminal PDC-109 protein: an overview of biochemical and functional properties.

PubMed

Srivastava, N; Jerome, A; Srivastava, S K; Ghosh, S K; Kumar, Amit

2013-04-01

Although long-term storage of bovine semen is desirable for wider use, successful cryopreservation depends on several factors, including various proteins present in seminal plasma. One such group of proteins, viz. bovine seminal plasma (BSP) proteins represents the major protein fraction in bovine seminal plasma. They constitute three major heparin-binding (HB-) acidic proteins secreted by seminal vesicles, viz. BSP-A1/-A2 (PDC-109), BSP-A3 and BSP-30-kDa. By purification studies it was deduced that PDC-109 is a polypeptide of 109 amino acids and contains two tandem repeating fibronectin type-II (Fn-II) domains, preceded by a 23 residue N-terminal domain. Though BSP-A1 and BSP-A2 are biochemically similar they differ only in glycosylation and their mixture is called PDC-109 or gonadostatins. PDC-109 exists as a polydisperse, multimeric self-associated molecule and possesses multifunctional properties, viz. binding to the surface of plasma membrane of spermatozoa causing conformational change in the sperm surface proteins and enhances motility. Besides binding, PDC-109 protein provokes cholesterol efflux from sperm membrane and promotes sperm reservoir by interacting with oviductal membrane. Interaction of sperm with PDC-109 protein induces sperm capacitation and acrosome reaction. However, prolonged exposure of spermatozoa with free floating PDC-109 protein as during processing for preservation, increases cholesterol efflux from spermatozoa. The efflux of sperm membrane cholesterol and disturbance in cholesterol:phospholipids ratio causes destabilization of plasma membrane thereby inducing cryoinjury to the sperm. In this review, the biochemical, functional properties of PDC-109 protein and its role during semen cryopreservation is summarized. Copyright © 2013 Elsevier B.V. All rights reserved.

Tools to evaluate the conformation of protein products.

PubMed

Manta, Bruno; Obal, Gonzalo; Ricciardi, Alejandro; Pritsch, Otto; Denicola, Ana

2011-06-01

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the correct conformation of the produced protein is crucial. There is no single method to assess every aspect of protein structure or function. Depending on the protein, the methods of choice vary. There are general methods to evaluate not only mass and primary sequence of the protein, but also higher-order structure. This review outlines the principal techniques for determining the conformation of a protein from structural (biophysical methods) to functional (in vitro binding assays) analyses. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

PubMed

Camadro, J M; Matringe, M; Thome, F; Brouillet, N; Mornet, R; Labbe, P

1995-05-01

Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

PubMed Central

Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

1996-01-01

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

PubMed

Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

1996-01-01

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

PubMed

Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

2016-05-16

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

Two novel genes, fanA and fanB, involved in the biogenesis of K99 fimbriae.

PubMed

Roosendaal, E; Boots, M; de Graaf, F K

1987-08-11

The nucleotide sequence of the region located transcriptionally upstream of the K99 fimbrial subunit gene (fanC) was determined. Several putative transcription signals and two open reading frames, designated fanA and fanB, became apparent. Frameshift mutations in fanA and fanB reduced K99 fimbriae expression 8-fold and 16-fold, respectively. Complementation of the mutants in trans restored the K99 expression to about 75% of the wild type level, indicating that fanA and fanB code for transacting polypeptides involved in the biogenesis of K99 fimbriae. The fanA and fanB gene products FanA and FanB were not detectable in minicell preparations, indicating that both polypeptides are synthesized in very small amounts. However, in an in vitro DNA directed translation system FanA and FanB could be identified. The deduced amino acid sequences of FanA and FanB showed that both polypeptides contain no signal peptides, indicating a cytoplasmic location. Furthermore, the polypeptides are very hydrophilic, mainly basic, and exhibit remarkable homology to each other and to a regulatory protein (papB) encoded by the pap-operon (1). Some of these features are characteristics of nucleic acid binding proteins, which suggests that FanA and FanB have a regulatory function in the synthesis of FanC and the auxiliary polypeptides FanD-H.

Identification of a major polypeptide of the nuclear pore complex

PubMed Central

1982-01-01

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes. PMID:7153248

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity*

PubMed Central

Liu, Bernard A.; Jablonowski, Karl; Shah, Eshana E.; Engelmann, Brett W.; Jones, Richard B.; Nash, Piers D.

2010-01-01

Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains. PMID:20627867

Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

PubMed

Nilsson, O Rickard; Lannergård, Jonas; Morgan, B Paul; Lindahl, Gunnar; Gustafsson, Mattias C U

2013-01-01

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

Novel sodium channel inhibitor from Conus geographus: purification, structure, and pharmacological properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanagawa, Y.; Abe, T.; Satake, M.

1988-08-23

A novel toxin, tentatively named conotoxin GS (CGS), has been isolated form a marine snail, Conus geographus. CGS was found to exist as a single polypeptide chain, consisting of 34 amino acid residues, cross-linked by three disulfide bonds. Its amino acid sequence was shown to be Ala-Cys-Ser-Gly-Arg-Gly-Ser-Arg-Cys-Hyp-Hyp-Gln-Cys-Cys-Met-Gly-Leu-Arg-Cys-Gly-Arg-Gly-Asn-Pro-Gln-Lys-Cys-Ile-Gly-Ala-His-Gla-Asp-Val. In competition experiments, CGS inhibited the bindings of (/sup 3/H)Lys-tetrodotoxin ((/sup 3/H)Lys-TTX) and (/sup 3/H)propionylconotoxin GIIIA to Electrophorus electricus electroplax membranes, with K/sub i/ values of 34 nM and 24 nM, respectively. The toxin inhibited the binding of (/sup 3/H)Lys-TTX (1 nM) to rat skeletal muscle homogenates with an IC/sub 50/ value ofmore » 880 nM but showed very little effect on this binding to the rat brain P/sub 2/ fraction at 10 ..mu..M. These binding studies indicate that CGS belongs to the same group of Na channel inhibitors as TTX, STX (saxitoxin), and ..mu..-conotoxins. Although CGS, like the ..mu..-conotoxins, is a pharmacological probe for distinguishing between neuronal and muscle Na channel subtypes, the homology in the sequences of CGS and ..mu..-conotoxins is very limited.« less

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp.

PubMed Central

Ruddock, L. W.; Freedman, R. B.; Klappa, P.

2000-01-01

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding. PMID:10794419

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.

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

2015-07-07

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.

Modeling the conformation of polyphenols and their complexation with polypeptides: self-association of catechin and its complexation with L-proline glycine oligomers.

Treesearch

Fred L. Tobiason; Richard W. Hemingway; Gerard Vergoten

1999-01-01

Over the past 10 years, several scientific thrusts have come together in the study of flavanoids that make it possible to move forward into the study of complexation between polyphenols and polypeptides. Enhanced understanding of the conformational properties of flavanoid monomers and polyflavanoids through...

Avian pathogenic Escherichia coli bind fibronectin and laminin.

PubMed

Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

2009-04-01

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain

DOE PAGES

Wu, R.; Wilton, R.; Cuff, M. E.; ...

2017-02-07

The tandem Per-Arnt-Sim (PAS) like sensors are commonly found in signal transduction proteins. The periplasmic solute binding protein (SBP) domains are found ubiquitously and are generally involved in solute transport. These domains are widely observed as parts of separate proteins but not within the same polypeptide chain. We report the structural and biochemical characterization of the extracellular ligand-binding receptor, Dret_0059 from Desulfohalobium retbaense DSM 5692, an organism isolated from the Retba salt lake in Senegal. The structure of Dret_0059 consists of a novel combination of SBP and TPAS sensor domains. The N-terminal region forms an SBP domain and the C-terminalmore » region folds into a tandem PAS-like domain structure. A ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS domain of the TPAS. The differential scanning flourimetry studies in solution support the ligands observed in the crystal structure. There are only two other proteins with this structural architecture in the non-redundant sequence data base and we predict that they too bind the same substrates. There is significant interaction between the SBP and TPAS domains, and it is quite conceivable that the binding of one ligand will have an effect on the binding of the other. Our attempts to remove the ligands bound to the protein during expression were not successful, therefore, it is not clear what the relative affects are. The genomic context of this receptor does not contain any protein components expected for transport function, hence, we suggest that Dret_0059 is likely involved in signal transduction and not in solute transport.« less

Molecular Basis of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels

DTIC Science & Technology

1988-10-20

channels bind a scorpion toxins and sea anemone toxins, which act at an extracellular site and spccifically slow Na+ channel inactivation (Catterall, 1980...the molecule by antibodies as well as by polypeptide neurotoxins from scorpions, sea anemones , coral and snail (Catterall, 1980; Strichartz et al

Identification and quantification of the rat hepatocyte asialoglycoprotein receptor.

PubMed Central

Schwartz, A L; Marshak-Rothstein, A; Rup, D; Lodish, H F

1981-01-01

The asialoglycoprotein receptor from rat liver was purified by solubilization and affinity chromatography on asialoorosomucoid-Sepharose. The preparation yielded four distinct polypeptides of Mr 40,000-120,000. We prepared a monoclonal antibody that both immunoprecipitates solubilized receptor activity and blocks the binding of galactose-terminal glycoproteins to immobilized receptor. The monoclonal antibody and a rabbit antireceptor antiserum immunoprecipitated all four polypeptide species. Peptide analysis by two-dimensional chromatography of the individual 125I-labeled species showed nearly identical patterns, which also suggested that the four polypeptides have a similar primary structure. To identify and quantitate the asialoglycoprotein receptor on the hepatocyte cell surface, intact cells were iodinated with lactoperoxidase, and the solubilized membranes were treated with antireceptor antibody. The Mr 55,000 and Mr 65,000 species were the major species found. Our results suggest that the Mr of the surface receptor is at least 55,000 and that it comprises between 1-2% of the iodinated hepatocyte surface protein. Images PMID:6267585

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide 1

PubMed Central

Lin, Chentao; Thomashow, Michael F.

1992-01-01

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the “boiling-stable” polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed. ImagesFigure 1Figure 4Figure 5Figure 6Figure 7 PMID:16668917

The diageotropica mutant of tomato lacks high specific activity auxin binding sites

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rayle, D. L.; Lomax, T. L.

1989-01-01

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

PubMed

Fulcher, Luke J; Hutchinson, Luke D; Macartney, Thomas J; Turnbull, Craig; Sapkota, Gopal P

2017-05-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. © 2017 The Authors.

Uncoordinated (UNC)119: coordinating the trafficking of myristoylated proteins.

PubMed

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in Caenorhabditis elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking ofmyristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transport myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

Uncoordinated (UNC)119: Coordinating the Trafficking of Myristoylated Proteins

PubMed Central

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D.; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in C. elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking of myristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transpot myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. PMID:23000199

A further insight into the adsorption mechanism of protein on hydroxyapatite by FTIR-ATR spectrometry

NASA Astrophysics Data System (ADS)

Lin, Zhongyu; Hu, Ren; Zhou, Jianzhang; Ye, Yiwen; Xu, Zhaoxi; Lin, Changjian

2017-02-01

The adsorption mechanism of bovine serum albumin (BSA) on hydroxyapatite (HA) for different time intervals has been studied by Fourier transform infrared (FTIR)-attenuated total internal reflectance (ATR) spectrometry in this paper. The difference spectra obtained in HA and BSA frequency regions demonstrate that the binding of Pdbnd O, from the phosphate (PO43 -) of HA, to the hydrogen of methyl (- CH3), methene (- CH2) and amideII (- CNH) in the protein appears to be much faster and stronger than that of the Psbnd O group. In addition, Ca2 + must serve as a key role in the interaction of BSA with HA. The binding of Ca2 + to the oxygen of the peptide bond seems to induce a significant reconformation of polypeptide backbones from β-pleated sheet to α-helix and β-turn of helical circles. This alteration seems to have been accompanied by much hydrogen of polypeptides driven to bind PO43 - and OH- of the HA actively and much -C = O and Hsbnd Nsbnd groups of the peptide bond freed from inter-chain hydrogen bonding to react on Ca2 + and combine strongly with the HA surface. This might be well expected to promote the HA biomineralization.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system

PubMed Central

Fulcher, Luke J.; Hutchinson, Luke D.; Macartney, Thomas J.; Turnbull, Craig

2017-01-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. PMID:28490657

Cryo-Electron Microscopy Reconstruction Shows Poliovirus 135S Particles Poised for Membrane Interaction and RNA Release

PubMed Central

Butan, Carmen; Filman, David J.

2014-01-01

During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN. Improved rigid-body positioning of major capsid proteins established reliably which polypeptide segments become disordered or rearranged. The virus-to-135S transition includes expansion of 4%, rearrangements of the GH loops of VP3 and VP1, and disordering of C-terminal extensions of VP1 and VP2. The N terminus of VP1 rearranges to become externalized near its quasi-3-fold exit, binds to rearranged GH loops of VP3 and VP1, and attaches to the top surface of VP2. These details improve our understanding of subsequent stages of infection, including endocytosis and RNA transfer into the cytoplasm. PMID:24257617

Purification and characterization of the glycogen-bound protein phosphatase from rat liver.

PubMed

Wera, S; Bollen, M; Stalmans, W

1991-01-05

Glycogen-bound protein phosphatase G from rat liver was transferred from glycogen to beta-cyclodextrin (cycloheptaamylose) linked to Sepharose 6B. After removal of the catalytic subunit and of contaminating proteins with 2 M NaCl, elution with beta-cyclodextrin yielded a single protein on native polyacrylamide gel electrophoresis and two polypeptides (161 and 54 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several lines of evidence indicate that the latter polypeptides are subunits of the protein phosphatase G holoenzyme. First, these polypeptides were also present, together with the catalytic subunit, in the extensively purified holoenzyme. Also, polyclonal antibodies against these polypeptides were able to bind the holoenzyme. Further, while bound to cyclodextrin-Sepharose, the polypeptides were able to recombine with separately purified type-1 (AMD) catalytic subunit, but not with type-2A (PCS) catalytic subunit. The characteristics of the reconstituted enzyme resembled those of the nonpurified protein phosphatase G. At low dilutions, the spontaneous phosphorylase phosphatase activity of the reconstituted enzyme was about 10 times lower than that of the catalytic subunit, but it was about 1000-fold more resistant to inhibition by the modulator protein (inhibitor-2). In contrast with the free catalytic subunit, the reconstituted enzyme co-sedimented with glycogen, and it was able to activate purified liver glycogen synthase b. Also, the synthase phosphatase activity was synergistically increased by a cytosolic phosphatase and inhibited by physiological concentrations of phosphorylase alpha and of Ca2+.

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

PubMed Central

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the conformational properties of unfolded states in water and in influencing chain expansion upon denaturation. PMID:25664638

Zonadhesin D3-Polypeptides Vary among Species but Are Similar in Equus Species Capable of Interbreeding1

PubMed Central

Tardif, Steve; Brady, Heidi A.; Breazeale, Kelly R.; Bi, Ming; Thompson, Leslie D.; Bruemmer, Jason E.; Bailey, Laura B.; Hardy, Daniel M.

2009-01-01

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%–72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed. PMID:19794156

Amino acid sequence of the human fibronectin receptor

PubMed Central

1987-01-01

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+- binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors. PMID:2958481

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bayer, R.

1990-03-06

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

Heat-induced formation of a specific binding site for self-assembled Congo Red in the V domain of immunoglobulin L chain lambda.

PubMed

Piekarska, B; Konieczny, L; Rybarska, J; Stopa, B; Zemanek, G; Szneler, E; Król, M; Nowak, M; Roterman, I

2001-11-01

Moderate heating (40-50 degrees C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon-like micellar bodies. The L chain lambda dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye-protein (L lambda chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40-45 degrees C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55 degrees C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL-VL domain incoherence, and protein melting. Of these three possibilities, local low-energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N-terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19-amino acid N-terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye-protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74-80 and 105-110, as shown by model analysis. The character of the temperature-induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed. Copyright 2001 John Wiley & Sons, Inc.

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Q. X. H.; Dementieva, I. S. D.; Walsh, M. A. W.

2001-02-23

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom}more » based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.« less

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rollins, T.E.; Siciliano, S.; Kobayashi, S.

1991-02-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42,more » 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.« less

Molecular properties of food allergens.

PubMed

Breiteneder, Heimo; Mills, E N Clare

2005-01-01

Plant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators.

Evolution of heliobacteria: implications for photosynthetic reaction center complexes

NASA Technical Reports Server (NTRS)

Vermaas, W. F.; Blankenship, R. E. (Principal Investigator)

1994-01-01

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein show rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to the core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

Deep-etch visualization of proteins involved in clathrin assembly

PubMed Central

1988-01-01

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined. PMID:3417785

Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

PubMed Central

Xu, Zhi-Xue; Zhang, Qiang; Ma, Gong-Li; Chen, Cong-Heng; He, Yan-Ming; Xu, Li-Hui; Zhang, Yuan; Zhou, Guang-Rong; Li, Zhen-Hua

2016-01-01

The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−)-epigallocatechin gallate (EGCG) is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III)/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III) and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III) and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III) could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III)/EGCG complex in molar ratio of 1 : 1 as Al(EGCG)(H2O)2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes. PMID:28074190

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

PubMed Central

Yan, Huan; Zhong, Guocai; Xu, Guangwei; He, Wenhui; Jing, Zhiyi; Gao, Zhenchao; Huang, Yi; Qi, Yonghe; Peng, Bo; Wang, Haimin; Fu, Liran; Song, Mei; Chen, Pan; Gao, Wenqing; Ren, Bijie; Sun, Yinyan; Cai, Tao; Feng, Xiaofeng; Sui, Jianhua; Li, Wenhui

2012-01-01

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI: http://dx.doi.org/10.7554/eLife.00049.001 PMID:23150796

A novel photoaffinity ligand for the phencyclidine site of the N-methyl-D-aspartate receptor labels a Mr 120,000 polypeptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonders, M.S.; Barmettler, P.; Lee, J.A.

1990-04-25

A radiolabeled photoaffinity ligand has been developed for the N-methyl-D-aspartate (NMDA)-preferring excitatory amino acid receptor complex. (3H)3-Azido-(5S, 10R)(+)-5-methyl-10,11-dihydro-5H- dibenzo(a,d)cyclohepten-5,10-imine (3H)3-azido-MK-801 demonstrated nearly identical affinity, density of binding sites, selectivity, pH sensitivity, and pharmacological profile in reversible binding assays with guinea pig brain homogenates to those displayed by its parent compound, MK-801. When employed in a photo-labeling protocol designed to optimize specific incorporation, (3H)3-azido-MK-801 labeled a single protein band which migrated in sodium dodecyl sulfate-polyacrylamide gels with Mr = 120,000. Incorporation of tritium into this band was completely inhibited when homogenates and (3H)3-azido-MK-801 were coincubated with 10 microM phencyclidine. These datamore » suggest that the phencyclidine site of the NMDA receptor complex is at least in part comprised of a Mr = 120,000 polypeptide.« less

Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?

PubMed

Abdullahi, Wazir; Davis, Thomas P; Ronaldson, Patrick T

2017-07-01

Drug delivery to the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-β signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.

Component effect of stem cell-loaded thermosensitive polypeptide hydrogels on cartilage repair.

PubMed

Liu, He; Cheng, Yilong; Chen, Jinjin; Chang, Fei; Wang, Jincheng; Ding, Jianxun; Chen, Xuesi

2018-06-01

Biophysical properties of the desired biomimetic scaffolds, such as porosity and elasticity, have been proven associated with the efficacy of cartilage regeneration. In this work, the copolymers of poly(l-alanine)-block-poly(ethylene glycol)-block-poly(l-alanine) (PA-PEG-PA) and poly(l-alanine-co-l-phenylalanine)-block-poly(ethylene glycol)-block-poly(l-alanine-co-l-phenylalanine) (PAF-PEG-PAF) with different ratios of alanine to phenylalanine were synthesized. The introduction of a hydrophobic amino acid, i.e., phenylalanine, into polyalanine-based thermosensitive hydrogel led to the enhanced gelation behaviors and upregulated mechanical properties. Moreover, the increase of phenylalanine content resulted in the enlarged pore size and enhanced mechanical strength of PAF-PEG-PAF thermogel, followed by the regeneration of hyaline-like cartilage with reduced fibrous tissue formation in vivo. The findings indicated the great potential of thermosensitive polypeptide hydrogels in cartilage tissue engineering. Articular cartilage defect has limited self-repair ability due to the lack of blood supply and innervation, which may lead to knee osteoarthritis afterwards. Injectable hydrogels are demonstrated possessing outstanding properties as biomimetic scaffolds in cartilage tissue engineering, while the effect of biophysical properties on the efficacy of cartilage regeneration has not been revealed. Herein, the poly(ethylene glycol)-polypeptide triblock copolymers with different ratios of alanine to phenylalanine were synthesized. The sol-to-gel transition temperature and the critical gelation concentration decreased as the increased amount of phenylalanine unit, resulting in the enlarged pore size and enhanced mechanical strength. These features lead to better regeneration of hyaline-like cartilage with reduced fibrous tissue formation, indicating great potential of thermosensitive polypeptide hydrogels for efficient cartilage repair. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

PubMed

Muiznieks, Lisa D; Reichheld, Sean E; Sitarz, Eva E; Miao, Ming; Keeley, Fred W

2015-10-01

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties. © 2015 Wiley Periodicals, Inc.

Identification of the bombesin receptor on murine and human cells by cross-linking experiments.

PubMed

Kris, R M; Hazan, R; Villines, J; Moody, T W; Schlessinger, J

1987-08-15

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.

Structural basis of substrate specificity in the serine proteases.

PubMed Central

Perona, J. J.; Craik, C. S.

1995-01-01

Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518

Modeling the Embrace of a Mutator: APOBEC Selection of Nucleic Acid Ligands.

PubMed

Salter, Jason D; Smith, Harold C

2018-05-23

The 11-member APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of zinc-dependent cytidine deaminases bind to RNA and single-stranded DNA (ssDNA) and, in specific contexts, modify select (deoxy)cytidines to (deoxy)uridines. In this review, we describe advances made through high-resolution co-crystal structures of APOBECs bound to mono- or oligonucleotides that reveal potential substrate-specific binding sites at the active site and non-sequence-specific nucleic acid binding sites distal to the active site. We also discuss the effect of APOBEC oligomerization on functionality. Future structural studies will need to address how ssDNA binding away from the active site may enhance catalysis and the mechanism by which RNA binding may modulate catalytic activity on ssDNA. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

PubMed Central

Kalidass, Bhagyalakshmi; Ul-Haque, Muhammad Farhan; Baral, Bipin S.; DiSpirito, Alan A.

2014-01-01

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin. PMID:25416758

Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor.

PubMed

Nkongolo, Shirin; Ni, Yi; Lempp, Florian A; Kaufman, Christina; Lindner, Thomas; Esser-Nobis, Katharina; Lohmann, Volker; Mier, Walter; Mehrle, Stefan; Urban, Stephan

2014-04-01

Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection. HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding. Cyclosporin A inhibited hepatitis B and D virus infections during and--less pronounced--prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8 μM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive. HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Acquisition of haemoglobin-bound iron by strains of the Actinobacillus minor/"porcitonsillarum" complex.

PubMed

Arya, Gitanjali; Niven, Donald F

2011-03-24

Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of ∼ 72 and ∼ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of ∼ 65 and ∼ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated. Copyright © 2010 Elsevier B.V. All rights reserved.

Photoaffinity labeling the substance P receptor using a derivative of substance P containing para-benzoylphenylalanine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyd, N.D.; White, C.F.; Leeman, S.E.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine (L-Phe(pBz)) in place of the Phe{sup 8} residue of SP. (Phe{sup 8}(OpBz))SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the {sup 125}I-labeled Bolton-Hunter conjugate of (Phe{sup 8}(pBz))SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites with an affinity K{sub D} = 0.4 nM. The binding of {sup 125}I-(Phe{sup 8}(pBz))SP was inhibited competitivelymore » by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70{percent} of the specifically bound {sup 125}I-(Phe{sup 8}(pBz))SP underwent covalent linkage to two polypeptides of M{sub r} = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on {sup 125}I-(Phe{sup 8}(pBz))SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies. The highly specific and remarkably efficient photolabeling achieved with {sup 125}I-(Phe{sup 8}(pBz))SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor.« less

Competition between metals for binding to methanobactin enables expression of soluble methane monooxygenase in the presence of copper.

PubMed

Kalidass, Bhagyalakshmi; Ul-Haque, Muhammad Farhan; Baral, Bipin S; DiSpirito, Alan A; Semrau, Jeremy D

2015-02-01

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Zinc-mediated binding of a low-molecular-weight stabilizer of the host anti-viral factor apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G.

PubMed

Radwan, Mohamed O; Sonoda, Sachiko; Ejima, Tomohiko; Tanaka, Ayumi; Koga, Ryoko; Okamoto, Yoshinari; Fujita, Mikako; Otsuka, Masami

2016-09-15

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Surface active complexes formed between keratin polypeptides and ionic surfactants.

PubMed

Pan, Fang; Lu, Zhiming; Tucker, Ian; Hosking, Sarah; Petkov, Jordan; Lu, Jian R

2016-12-15

Keratins are a group of important proteins in skin and hair and as biomaterials they can provide desirable properties such as strength, biocompatibility, and moisture regaining and retaining. The aim of this work is to develop water-soluble keratin polypeptides from sheep wool and then explore how their surface adsorption behaves with and without surfactants. Successful preparation of keratin samples was demonstrated by identification of the key components from gel electrophoresis and the reproducible production of gram scale samples with and without SDS (sodium dodecylsulphate) during wool fibre dissolution. SDS micelles could reduce the formation of disulphide bonds between keratins during extraction, reducing inter-molecular crosslinking and improving keratin polypeptide solubility. However, Zeta potential measurements of the two polypeptide batches demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing complete removal of SDS during purification by dialysis. In spite of different solubility from the two batches of keratin samples prepared, very similar adsorption and aggregation behavior was revealed from surface tension measurements and dynamic light scattering. Mixing of keratin polypeptides with SDS and C 12 TAB (dodecyltrimethylammonium bromide) led to the formation of keratin-surfactant complexes that were substantially more effective at reducing surface tension than the polypeptides alone, showing great promise in the delivery of keratin polypeptides via the surface active complexes. Neutron reflection measurements revealed the coexistence of surfactant and keratin polypeptides at the interface, thus providing the structural support to the observed surface tension changes associated with the formation of the surface active complexes. Copyright © 2016. Published by Elsevier Inc.

Can misfolded proteins be beneficial? The HAMLET case.

PubMed

Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina

2009-01-01

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

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.

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

PubMed

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 (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.

Interaction of β-sheet folds with a gold surface.

PubMed

Hoefling, Martin; Monti, Susanna; Corni, Stefano; Gottschalk, Kay Eberhard

2011-01-01

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance.

Probing the organic-mineral interface at the molecular level in model biominerals.

PubMed

Metzler, Rebecca A; Kim, Il Won; Delak, Katya; Evans, John Spencer; Zhou, Dong; Beniash, Elia; Wilt, Fred; Abrecht, Mike; Chiou, Jau-Wern; Guo, Jinghua; Coppersmith, Susan N; Gilbert, P U P A

2008-03-18

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners.

PubMed

Jiang, Bingbing; Defusco, Elizabeth; Li, Bingyun

2010-12-13

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.

Polypeptide Multilayer Film Co-Delivers Oppositely-Charged Drug Molecules in Sustained Manners

PubMed Central

Jiang, Bingbing; DeFusco, Elizabeth; Li, Bingyun

2010-01-01

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intending for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely-charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO3) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO3 templates. Two oppositely-charged drugs were loaded into capsules within polypeptide multilayer films post-preparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g. opposite charges) any time post-preparation (e.g. minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely-charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved. PMID:21058719

Design and synthesis of nonionic copolypeptide hydrogels with reversible thermoresponsive and tunable physical properties.

PubMed

Zhang, Shanshan; Alvarez, Daniel J; Sofroniew, Michael V; Deming, Timothy J

2015-04-13

Polypeptide-based formulations that undergo liquid to hydrogel transitions upon change in temperature have become desirable targets since they can be mixed with cells or injected into tissues as liquids, and subsequently transform into rigid scaffolds or depots. Such materials have been challenging to prepare using synthetic polypeptides, especially when reversible gelation and tunable physical properties are desired. Here, we designed and prepared new nonionic diblock copolypeptide hydrogels (DCH) containing hydrophilic poly(γ-[2-(2-methoxyethoxy)ethyl]-rac-glutamate) and hydrophobic poly(l-leucine) segments, named DCHEO, and also further incorporated copolypeptide domains into DCHEO to yield unprecedented thermoresponsive DCH, named DCHT. Although previous attempts to prepare nonionic hydrogels composed solely of synthetic polypeptides have been unsuccessful, our designs yielded materials with highly reversible thermal transitions and tunable properties. Nonionic, thermoresponsive DCHT were found to support the viability of suspended mesenchymal stem cells in vitro and were able to dissolve and provide prolonged release of both hydrophilic and hydrophobic molecules. The versatility of these materials was further demonstrated by the independent molecular tuning of DCHT liquid viscosity at room temperature and DCHT hydrogel stiffness at elevated temperature, as well as the DCHT liquid to hydrogel transition temperature itself.

Proline-Rich Salivary Proteins Have Extended Conformations

PubMed Central

Boze, Hélène; Marlin, Thérèse; Durand, Dominique; Pérez, Javier; Vernhet, Aude; Canon, Francis; Sarni-Manchado, Pascale; Cheynier, Véronique; Cabane, Bernard

2010-01-01

Abstract Three basic proline-rich salivary proteins have been produced through the recombinant route. IB5 is a small basic proline-rich protein that is involved in the binding of plant tannins in the oral cavity. II-1 is a larger protein with a closely related backbone; it is glycosylated, and it is also able to bind plant tannins. II-1ng has the same polypeptidic backbone as II-1, but it is not glycosylated. Small angle x-ray scattering experiments on dilute solutions of these proteins confirm that they are intrinsically disordered. IB5 and II-1ng can be described through a chain model including a persistence length and cross section. The measured radii of gyration (Rg = 27.9 and 41.0 ± 1 Å respectively) and largest distances (rmax = 110 and 155 ± 10 Å respectively) show that their average conformations are rather extended. The length of the statistical segment (twice the persistence length) is b = 30 Å, which is larger than the usual value (18 Å − 20 Å) for unstructured polypeptide chains. These characteristics are presumably related to the presence of polyproline helices within the polypeptidic backbones. For both proteins, the radius of gyration of the chain cross-section is Rc = 2.7 ± 0.2Å. The glycosylated protein II-1 has similar conformations but the presence of large polyoside sidegroups yields the structure of a branched macromolecule with the same hydrophobic backbone and hydrophilic branches. It is proposed that the unusually extended conformations of these proteins in solution facilitate the capture of plant tannins in the oral cavity. PMID:20643086

Repeats of base oligomers as the primordial coding sequences of the primeval earth and their vestiges in modern genes.

PubMed

Ohno, S

1984-01-01

Three outstanding properties uniquely qualify repeats of base oligomers as the primordial coding sequences of all polypeptide chains. First, when compared with randomly generated base sequences in general, they are more likely to have long open reading frames. Second, periodical polypeptide chains specified by such repeats are more likely to assume either alpha-helical or beta-sheet secondary structures than are polypeptide chains of random sequence. Third, provided that the number of bases in the oligomeric unit is not a multiple of 3, these internally repetitious coding sequences are impervious to randomly sustained base substitutions, deletions, and insertions. This is because the recurring periodicity of their polypeptide chains is given by three consecutive copies of the oligomeric unit translated in three different reading frames. Accordingly, when one reading frame is open, the other two are automatically open as well, all three being capable of coding for polypeptide chains of identical periodicity. Under this circumstance, a frame shift due to the deletion or insertion of a number of bases that is not a multiple of 3 fails to alter the down-stream amino acid sequence, and even a base change causing premature chain-termination can silence only one of the three potential coding units. Newly arisen coding sequences in modern organisms are oligomeric repeats, and most of the older genes retain various vestiges of their original internal repetitions. Some of the genes (e.g., oncogenes) have even inherited the property of being impervious to randomly sustained base changes.

Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.

PubMed

Macnaughton, T B; Gowans, E J; Reinboth, B; Jilbert, A R; Burrell, C J

1990-06-01

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

The chemical and dynamical influence of the anti-viral drug amantadine on the M2 proton channel transmembrane domain.

PubMed

Hu, Jun; Fu, Riqiang; Cross, Timothy A

2007-07-01

The M(2) proton channel plays a vital role in the life cycle of the influenza A virus. His(37), the key residue in the M(2) transmembrane domain (M(2)-TMD), plays a central role in the proton conductance mechanism. The anti-influenza drug, amantadine, inhibits the channel activity through binding to the pore of the M(2) channel. The nuclear spin relaxation data and polarization inversion spin exchange at the magic angle spectra show that both the polypeptide backbone and His(37) side chain are more constrained in the presence of amantadine. Using (15)N cross polarization magic-angle spinning NMR spectroscopy, the protonation of His(37) of M(2)-TMD in lipid bilayers was monitored in the absence and presence of amantadine as a function of pH. Binding amantadine lowers the His(37) pK(a) values by approximately three orders of magnitude compared with the first pK(a) of histidine in amantadine-free M(2)-TMD. Amantadine's influence on the His(37) chemical properties suggests a novel mechanism by which amantadine may inhibit proton conductance.

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics

PubMed Central

Abedini, Andisheh; Plesner, Annette; Cao, Ping; Ridgway, Zachary; Zhang, Jinghua; Tu, Ling-Hsien; Middleton, Chris T; Chao, Brian; Sartori, Daniel J; Meng, Fanling; Wang, Hui; Wong, Amy G; Zanni, Martin T; Verchere, C Bruce; Raleigh, Daniel P; Schmidt, Ann Marie

2016-01-01

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death. DOI: http://dx.doi.org/10.7554/eLife.12977.001 PMID:27213520

Purification and biophysical characterization of the core protease domain of anthrax lethal factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gkazonis, Petros V.; Dalkas, Georgios A.; Chasapis, Christos T.

2010-06-04

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn{sup 2+} binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site aremore » essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF{sub 672-776}) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ({sup 1}H, {sup 13}C, {sup 15}N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn{sup 2+}, suitable for high resolution structural analysis by NMR.« less

Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/β.

PubMed

Honda, Akinobu; Chigwechokha, Petros Kingstone; Kamada-Futagami, Yuko; Komatsu, Masaharu; Shiozaki, Kazuhiro

2018-06-01

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin β and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Yeongseon; Choi, Won Tae; Heller, William T.

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE PAGES

Jang, Yeongseon; Choi, Won Tae; Heller, William T.; ...

2017-07-27

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

A Native to Amyloidogenic Transition Regulated by a Backbone Trigger

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eakin,C.; Berman, A.; Miranker, A.

2006-01-01

Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by beta-2-microglobulin (beta2m) in dialysis-related amyloidosis. Access of beta2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a beta2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamicmore » and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type beta2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.« less

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-11-11

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed Central

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-01-01

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

The functional characterization and comparison of two single CRD containing C-type lectins with novel and typical key motifs from Portunus trituberculatus.

PubMed

Huang, Mengmeng; Mu, Changkao; Wu, Yuehong; Ye, Fei; Wang, Dan; Sun, Cong; Lv, Zhengbing; Han, Bingnan; Wang, Chunlin; Xu, Xue-Wei

2017-11-01

C-type lectins are a superfamily of Ca 2+ -dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads. Copyright © 2017 Elsevier Ltd. All rights reserved.

A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo.

PubMed

Turabee, Md Hasan; Thambi, Thavasyappan; Duong, Huu Thuy Trang; Jeong, Ji Hoon; Lee, Doo Sung

2018-02-27

Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 μg mL -1 ). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

PubMed Central

Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

1990-01-01

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.

PubMed

Davis, J Q; McLaughlin, T; Bennett, V

1993-04-01

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

PubMed Central

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-01-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result. PMID:7853501

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

PubMed

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-03-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zhuji; Chen, Chen; Barbieri, Joseph T.

2010-02-22

Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that ofmore » HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using {alpha}(1-3,4)-fucosidase, endo-{beta}-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain {alpha}2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.« less

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein.

PubMed

Yagi, Sota; Akanuma, Satoshi; Kaji, Asumi; Niiro, Hiroya; Akiyama, Hayato; Uchida, Tatsuya; Yamagishi, Akihiko

2018-02-01

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Identification of polypeptides necessary for chemotaxis in Escherichia coli.

PubMed Central

Silverman, M; Simon, M

1977-01-01

Molecular cloning techniques were used to construct Escherichia coli-lambda hybrids that contained many of the genes necessary for flagellar rotation and chemotaxis. The properties of specific hybrids that carried the classical "cheA" and "cheB" loci were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the hybrids redefined the "cheA" and "cheB" regions. Six genes were resolved: cheA, cheW, cheX, cheB, cheY, and cheZ. They directed the synthesis of specific polypeptides with the following apparent molecular weights: cheA, 76,000 and 66,000; cheW, 12,000; cheX, 28,000; cheB, 38,000; cheY, 8,000; and cheZ, 24,000. The presence of another gene, cheM, was inferred from the protein synthesis experiments. The cheM gene directed the synthesis of polypeptides with apparent molecular weights of 63,000, 61,000, and 60,000. The synthesis of all of these polypeptides is regulated by the same mechanisms that regulate the synthesis of flagellar-related structural components. Images PMID:324984

Protein amyloids develop an intrinsic fluorescence signature during aggregation†

PubMed Central

Chan, Fiona T. S.; Kaminski Schierle, Gabriele S.; Kumita, Janet R.; Bertoncini, Carlos W.; Dobson, Christopher M.; Kaminski, Clemens F.

2017-01-01

We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1–40) and (1–42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner. PMID:23420088

Multimodal switching of conformation and solubility in homocysteine derived polypeptides.

PubMed

Kramer, Jessica R; Deming, Timothy J

2014-04-16

We report the design and synthesis of poly(S-alkyl-L-homocysteine)s, which were found to be a new class of readily prepared, multiresponsive polymers that possess the unprecedented ability to respond in different ways to different stimuli, either through a change in chain conformation or in water solubility. The responsive properties of these materials are also effected under mild conditions and are completely reversible for all pathways. The key components of these polymers are the incorporation of water solubilizing alkyl functional groups that are integrated with precisely positioned, multiresponsive thioether linkages. This promising system allows multimodal switching of polypeptide properties to obtain desirable features, such as coupled responses to multiple external inputs.

In vitro myogenesis induced by human recombinant elastin-like proteins.

PubMed

D'Andrea, Paola; Scaini, Denis; Ulloa Severino, Luisa; Borelli, Violetta; Passamonti, Sabina; Lorenzon, Paola; Bandiera, Antonella

2015-10-01

Mammalian adult skeletal muscle has a limited ability to regenerate after injury, usage or trauma. A promising strategy for successful regenerative technology is the engineering of bio interfaces that mimic the characteristics of the extracellular matrix. Human elastin-like polypeptides (HELPs) have been synthesized as biomimetic materials that maintain some peculiar properties of the native protein. We developed a novel Human Elastin Like Polypeptide obtained by fusing the elastin-like backbone to a domain present in the α2 chain of type IV collagen, containing two RGD motives. We employed this peptide as adhesion substrate for C2C12 myoblasts and compared its effects to those induced by two other polypeptides of the HELP series. Myoblast adhered to all HELPs coatings, where they assumed morphology and cytoarchitecture that depended on the polypeptide structure. Adhesion to HELPs stimulated at a different extent cell proliferation and differentiation, the expression of Myosin Heavy Chain and the fusion of aligned fibers into multinucleated myotubes. Adhesion substrates significantly altered myotubes stiffness, measured by Atomic Force Microscopy, and differently affected the cells Ca(2+) handling capacity and the maturation of excitation-contraction coupling machinery, evaluated by Ca(2+) imaging. Overall, our findings indicate that the properties of HELP biopolymers can be exploited for dissecting the molecular connections underlying myogenic differentiation and for designing novel substrates for skeletal muscle regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antibodies to the Central Conserved Region of Respiratory Syncytial Virus (RSV) G Protein Block RSV G Protein CX3C-CX3CR1 Binding and Cross-Neutralize RSV A and B Strains0

PubMed Central

Choi, Youngjoo; Mason, Caleb S.; Jones, Les P.; Crabtree, Jackelyn; Jorquera, Patricia A.

2012-01-01

Abstract Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein–CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein–CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines. PMID:22551066

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

PubMed Central

ROZENFELD, P; DOCENA, G H; AÑÓN, M C; FOSSATI, C A

2002-01-01

Soy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific monoclonal antibodies were used in immunoblotting and competitive ELISA studies to identify a 30-kD component from soybean that cross-reacts with cow's milk caseins. Its IgE binding capacity was tested by EAST, employing sera from cow's milk allergic patients, not previously exposed to soy proteins. The 30 kD protein was isolated and partially sequenced. It is constituted by two polypeptides (A5 and B3) linked by a disulphide bond. The protein's capacity to bind to the different antibodies relies on the B3 poly-peptide. These results indicate that soy-based formula, which contains the A5-B3 glycinin molecule, could be involved in allergic reactions observed in cow's milk allergic patients exposed to soy-containing foods. PMID:12296853

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

PubMed Central

Ullers, Ronald S.; Houben, Edith N.G.; Raine, Amanda; ten Hagen-Jongman, Corinne M.; Ehrenberg, Måns; Brunner, Joseph; Oudega, Bauke; Harms, Nellie; Luirink, Joen

2003-01-01

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain. PMID:12756233

Interaction of β-Sheet Folds with a Gold Surface

PubMed Central

Hoefling, Martin; Monti, Susanna; Corni, Stefano; Gottschalk, Kay Eberhard

2011-01-01

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance. PMID:21687744

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Metal Ion-Induced Self-Assembly of a Multi-Responsive Block Copolypeptide into Well-Defined Nanocapsules.

PubMed

van Eldijk, Mark B; Schoonen, Lise; Cornelissen, Jeroen J L M; Nolte, Roeland J M; van Hest, Jan C M

2016-05-01

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule-forming designer polypeptides with a tailor-made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine-tag, a stimulus-responsive elastin-like polypeptide block, and a pH-responsive morphology-controlling viral capsid protein is presented. The self-assembly of this multi-responsive protein-based block copolymer is triggered by the addition of divalent metal ions. This assembly process yields monodisperse nanocapsules with a 20 nm diameter composed of 60 polypeptides. The well-defined nanoparticles are the result of the emergent properties of all the blocks of the polypeptide. These results demonstrate the feasibility of hexahistidine-tags to function as supramolecular cross-linkers. Furthermore, their potential for the metal ion-mediated encapsulation of hexahistidine-tagged proteins is shown. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dissipation at the angstrom scale: Probing the surface and interior of an enzyme

NASA Astrophysics Data System (ADS)

Alavi, Zahra; Zocchi, Giovanni

2018-05-01

Pursuing a materials science approach to understanding the deformability of enzymes, we introduce measurements of the phase of the mechanical response function within the nanorheology paradigm. Driven conformational motion of the enzyme is dissipative as characterized by the phase measurements. The dissipation originates both from the surface hydration layer and the interior of the molecule, probed by examining the effect of point mutations on the mechanics. We also document changes in the mechanics of the enzyme examined, guanylate kinase, upon binding its four substrates. GMP binding stiffens the molecule, ATP and ADP binding softens it, while there is no clear mechanical signature of GDP binding. A hyperactive two-Gly mutant is found to possibly trade specificity for speed. Global deformations of enzymes are shown to be dependent on both hydration layer and polypeptide chain dynamics.

Binding of Soluble Natural Ligands to a Soluble Human T-Cell Receptor Fragment Produced in Escherichia coli

NASA Astrophysics Data System (ADS)

Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.

1994-09-01

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

DOEpatents

Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

2007-03-13

The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

Subunit assembly of hemoglobin: an important determinant of hematologic phenotype.

PubMed

Bunn, H F

1987-01-01

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

PubMed Central

Hase, Manuela E.; Kuznetsov, Nikolai V.; Cordes, Volker C.

2001-01-01

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil–forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent α-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ∼7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed. PMID:11514627

Interaction of the Transactivation Domain of B-Myb with the TAZ2 Domain of the Coactivator p300: Molecular Features and Properties of the Complex

PubMed Central

Oka, Ojore; Waters, Lorna C.; Strong, Sarah L.; Dosanjh, Nuvjeevan S.; Veverka, Vaclav; Muskett, Frederick W.; Renshaw, Philip S.; Klempnauer, Karl-Heinz; Carr, Mark D.

2012-01-01

The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (Kd ∼1.0–10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (∼1200 Å2). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2. PMID:23300815

Construction of ultrasonic nanobubbles carrying CAIX polypeptides to target carcinoma cells derived from various organs.

PubMed

Zhu, Lianhua; Guo, Yanli; Wang, Luofu; Fan, Xiaozhou; Xiong, Xingyu; Fang, Kejing; Xu, Dan

2017-09-29

Ultrasound molecular imaging is a novel diagnostic approach for tumors, whose key link is the construction of targeted ultrasound contrast agents. However, available targeted ultrasound contrast agents for molecular imaging of tumors are only achieving imaging in blood pool or one type tumor. No targeted ultrasound contrast agents have realized targeted ultrasound molecular imaging of tumor parenchymal cells in a variety of solid tumors so far. Carbonic anhydrase IX (CAIX) is highly expressed on cell membranes of various malignant solid tumors, so it's a good target for ultrasound molecular imaging. Here, targeted nanobubbles carrying CAIX polypeptides for targeted binding to a variety of malignant tumors were constructed, and targeted binding ability and ultrasound imaging effect in different types of tumors were evaluated. The mean diameter of lipid targeted nanobubbles was (503.7 ± 78.47) nm, and the polypeptides evenly distributed on the surfaces of targeted nanobubbles, which possessed the advantages of homogenous particle size, high stability, and good safety. Targeted nanobubbles could gather around CAIX-positive cells (786-O and Hela cells), while they cannot gather around CAIX-negative cells (BxPC-3 cells) in vitro, and the affinity of targeted nanobubbles to CAIX-positive cells were significantly higher than that to CAIX-negative cells (P < 0.05). Peak intensity and duration time of targeted nanobubbles and blank nanobubbles were different in CAIX-positive transplanted tumor tissues in vivo (P < 0.05). Moreover, targeted nanobubbles in CAIX-positive transplanted tumor tissues produced higher peak intensity and longer duration time than those in CAIX-negative transplanted tumor tissues (P < 0.05). Finally, immunofluorescence not only confirmed targeted nanobubbles could pass through blood vessels to enter in tumor tissue spaces, but also clarified imaging differences of targeted nanobubbles in different types of transplanted tumor tissues. Targeted nanobubbles carrying CAIX polypeptides can specifically enhance ultrasound imaging in CAIX-positive transplanted tumor tissues and could potentially be used in early diagnosis of a variety of solid tumors derived from various organs.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma.

PubMed

Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

PubMed Central

Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function. PMID:29138803

An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

PubMed Central

Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

1998-01-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

PubMed

Bearden, S W; Staggs, T M; Perry, R D

1998-03-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Glycosphingolipid-facilitated membrane insertion and internalization of cobra cardiotoxin. The sulfatide.cardiotoxin complex structure in a membrane-like environment suggests a lipid-dependent cell-penetrating mechanism for membrane binding polypeptides.

PubMed

Wang, Chia-Hui; Liu, Jyung-Hurng; Lee, Shao-Chen; Hsiao, Chwan-Deng; Wu, Wen-Guey

2006-01-06

Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 angstroms resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.

A Conserved START Domain Coenzyme Q-binding Polypeptide is Required for Efficient Q Biosynthesis, Respiratory Electron Transport, and Antioxidant Function in Saccharomyces cerevisiae

PubMed Central

Morvaridi, Susan; Saiki, Ryoichi; Johnson, Jarrett S.; Liau, Wei-Siang; Hirano, Kathleen; Kawashima, Tadashi; Ji, Ziming; Loo, Joseph A.; Shepherd, Jennifer N.; Clarke, Catherine F.

2014-01-01

Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain. PMID:23270816

Coexpression patterns of vimentin and glial filament protein with cytokeratins in the normal, hyperplastic, and neoplastic breast.

PubMed Central

Gould, V. E.; Koukoulis, G. K.; Jansson, D. S.; Nagle, R. B.; Franke, W. W.; Moll, R.

1990-01-01

The authors studied by immunohistochemistry the intermediate filament (IF) protein profile of 66 frozen samples of breast tissue, including normal parenchyma, all variants of fibrocystic disease (FCD), fibroadenomas, cystosarcoma phylloides, and ductal and lobular carcinomas. Monoclonal antibodies (MAbs) to cytokeratins included MAb KA 1, which binds to polypeptide 5 in a complex with polypeptide 14 and recognizes preferentially myoepithelial cells; MAb KA4, which binds to polypeptides 14, 15, 16 and 19; individual MAbs to polypeptides 7, 13, and 16, 17, 18, and 19, and the MAb mixture AE1/AE3. The authors also applied three MAbs to vimentin (Vim), and three MAbs to glial filament protein (GFP). Selected samples were studied by double-label immunofluorescence microscopy and by staining sequential sections with some of the said MAbs, an MAb to alpha-smooth muscle actin, and well-characterized polyclonal antibodies for the possible coexpression of diverse types of cytoskeletal proteins. Gel electrophoresis and immunoblot analysis also were performed. All samples reacted for cytokeratins with MAbs AE1/AE3, although the reaction did not involve all cells. Monoclonal antibody KA4 stained preferentially the luminal-secretory cells in the normal breast and in FCD, whereas it stained the vast majority of cells in all carcinomas. Monoclonal antibody KA1 stained preferentially the basal-myoepithelial cells of the normal breast and FCD while staining tumor cell subpopulations in 4 of 31 carcinomas. Vimentin-positive cells were found in 8 of 12 normal breasts and in 12 of 20 FCD; in most cases, Vim-reactive cells appeared to be myoepithelial, but occasional luminal cells were also stained. Variable subpopulations of Vim-positive cells were noted in 9 of 20 ductal and in 1 of 7 lobular carcinomas. Glial filament protein-reactive cells were found in normal breast lobules and ducts and in 15 of 20 cases of FCD; with rare exceptions, GFP-reactivity was restricted to basally located, myoepithelial-appearing cells. Occasional GFP-reactive cells were found in 3 of 31 carcinomas. Evaluation of sequential sections and double-label immunofluorescence microscopy showed the coexpression of certain cytokeratins (possibly including polypeptides 14 and 17) with vimentin and alpha-smooth muscle actin together with GFP in some myoepithelial cells. The presence of GFP in myoepithelial cells was confirmed by gel electrophoresis and immunoblotting. Our results indicate that coexpression of cytokeratin with vimentin and/or GFP is comparatively frequent in normal basal-myoepithelial cells of the breast.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1700618

Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides.

PubMed

Sato, R; Oshio, H; Koike, H; Inoue, Y; Yoshida, S; Takahashi, N

1991-06-01

Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl beta-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein.

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species

PubMed Central

Liu, Liyun; Hastings, J. Woodland

2007-01-01

Noctiluca scintillans, a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size (≈100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca. PMID:17130452

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

PubMed

Zhang, Y; LeRoy, G; Seelig, H P; Lane, W S; Reinberg, D

1998-10-16

Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis.

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

PubMed Central

Aleksunes, Lauren M.

2010-01-01

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology. PMID:20103563

Silk as an innovative biomaterial for cancer therapy.

PubMed

Jastrzebska, Katarzyna; Kucharczyk, Kamil; Florczak, Anna; Dondajewska, Ewelina; Mackiewicz, Andrzej; Dams-Kozlowska, Hanna

2015-01-01

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

PubMed

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Fabrication of genetically engineered polypeptide@quantum dots hybrid nanogels for targeted imaging

NASA Astrophysics Data System (ADS)

Yang, Jie; Yao, Ming-Hao; Zhao, Dong-Hui; Zhang, Xiao-Shuai; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

2017-08-01

Nanogels have been widely used as multifunctional drug delivery carriers because of high water content, biocompatibility, and high loading capability. We designed and biosynthesized two triblock artificial polypeptides PC10A and PC10ARGD as vehicles for encapsulating hydrophobic materials. These polypeptides can form nanogels by self-assembly when the concentration is below 2% ( w/ v). The physical properties of nanogels, including size, surface potential, and targeting domain, are able to be tuned. Hydrophobic materials from molecular size to nano-size can be loaded into the polypeptide nanogels to form hybrid nanogels. Hydrophobic quantum dots CdSe@ZnS below 10 nM were loaded into the polypeptide nanogels by ultrasonic treatment. Encapsulation endows hydrophobic QDs with good tunability of size, water solubility, stability, targeting, and biocompatibility. PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels showed excellent biocompatibility, which the cellular viabilities of HeLa and MCF-7 cells treated with 1% PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels contained 20 nM QDs were above 90 and 80%, respectively. PC10ARGD@QDs hybrid nanogels with an arginine-glycine-aspartic acid motif present efficient receptor-mediated endocytosis in α v β 3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy. These results demonstrate that such polypeptide nanogels as nanocarriers are expected to have great potential applications in biomedicine.

Structural properties of pyruvate carboxylases from chicken liver and other sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barden, R.E.; Taylor, B.L.; Isohashi, F.

1975-11-01

Varieties of pyruvate carboxylase (pyruvate: CO/sub 2/ ligase (ADP-forming), EC 6.4.1.1) obtained from the livers of several species of vertebrates, including humans, all show the same basic structure. They are composed of large polypeptide chains of molecular weights ranging from 1.2 to 1.3 x 10/sup 5/ for the different varieties of the enzyme. The native form of the enzyme appears to be a tetramer with a molecular weight of about 5 x 10/sup 5/. In the case of pyruvate carboxylase from chicken liver each polypeptide chain contains a biotin moiety, thus supporting the thesis that the tetramer contains four identicalmore » polypeptide chains. Pyruvate carboxylase from yeast appears to be basically similar to those from the vertebrate species and has a tetrameric structure. Each protomer contains a single polypeptide chain with a molecular weight of 1.25 x 10/sup 5/. In contrast, pyruvate carboxylase from two bacterial species, Pseudomonas citronellolis and Azotobacter vinelandii, appears to be a dimer with a molecular weight (2.5 x 10/sup 5/) about half that of the animal and yeast species. As a further difference, each of the protomers of the bacterial enzymes contain two polypeptides of 6.5 and 5.4 x 10/sup 5/ molecular weight in the case of the Pseudomonas enzyme. The larger of the two polypeptides contains the biotin moiety. The functional units of the bacterial enzyme thus appear to contain two polypeptides while that of the liver and yeast enzymes is made up of a single chain. Neither of these arrangements corresponds with those of other biotin enzymes whose structure has been extensively studied (acetyl-CoA carboxylases from liver or Escherichia coli, and transcarboxylase from Propionibacterium). (auth)« less

Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers.

PubMed

Cho, S; Dong, S; Parent, K N; Chen, M

2016-01-01

Cytotoxic T lymphocyte (CTL) vaccine carriers are known to enhance the efficacy of vaccines, but a search for more effective carriers is warranted. Elastin-like polypeptides (ELPs) have been examined for many medical applications but not as CTL vaccine carriers. We aimed to create immune tolerant ELPs using a new polypeptide engineering practice and create CTL vaccine carriers using the ELPs. Four sets of novel ELPs, termed immune-tolerant elastin-like polypeptide (iTEP) were generated according to the principles dictating humoral immunogenicity of polypeptides and phase transition property of ELPs. The iTEPs were non-immunogenic in mice. Their phase transition feature was confirmed through a turbidity assay. An iTEP nanoparticle (NP) was assembled from an amphiphilic iTEP copolymer plus a CTL peptide vaccine, SIINFEKL. The NP facilitated the presentation of the vaccine by dendritic cells (DCs) and enhanced vaccine-induced CTL responses. A new ELP design and development practice was established. The non-canonical motif and the immune tolerant nature of the iTEPs broaden our insights about ELPs. ELPs, for the first time, were successfully used as carriers for CTL vaccines. It is feasible to concurrently engineer both immune-tolerant and functional peptide materials. ELPs are a promising type of CTL vaccine carriers.

Vicilin allergens of peanut and tree nuts (walnut, hazelnut and cashew nut) share structurally related IgE-binding epitopes.

PubMed

Barre, Annick; Sordet, Camille; Culerrier, Raphaël; Rancé, Fabienne; Didier, Alain; Rougé, Pierre

2008-03-01

Surface-exposed IgE-binding epitopes of close overall conformation were characterized on the molecular surface of three-dimensional models built for the vicilin allergens of peanut (Ara h 1), walnut (Jug r 2), hazelnut (Cor a 11) and cashew nut (Ana o 1). They correspond to linear stretches of conserved amino acid sequences mainly located along the C-terminus of the polypeptide chains. A glyco-epitope corresponding to an exposed N-glycosylation site could also interfere with the IgE-binding epitopes. All these epitopic regions should participate in the IgE-binding cross-reactivity commonly reported between tree nuts or between peanut and some tree nuts in sensitized individuals. Owing to this epitopic community which constitutes a risk of cross-sensitization, the avoidance or a restricted consumption of other tree nuts should be recommended to peanut-sensitized individuals.

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking

PubMed Central

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. PMID:22960045

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenman, W.M.; Meuser, R.U.

1986-02-01

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L/sub 2/ were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizingmore » activity. These data suggest that these putative chlamydial adhesions play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.« less

Antibody neutralization of retargeted measles viruses

PubMed Central

Lech, Patrycja J.; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J.; Nara, Peter L.; Russell, Stephen J.

2014-01-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

PubMed Central

Hansen, F G; Koefoed, S; Sørensen, L; Atlung, T

1987-01-01

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322. PMID:3034578

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

PubMed

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2002-10-01

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

[Engineered spider silk: the intelligent biomaterial of the future. Part I].

PubMed

Florczak, Anna; Piekoś, Konrad; Kaźmierska, Katarzyna; Mackiewicz, Andrzej; Dams-Kozłowska, Hanna

2011-06-17

The unique properties of spider silk such as strength, extensibility, toughness, biocompatibility and biodegradability are the reasons for the recent development in silk biomaterial technology. For a long time scientific progress was impeded by limited access to spider silk. However, the development of the molecular biology strategy was a breaking point in synthetic spider silk protein design. The sequences of engineered spider silk are based on the consensus motives of the corresponding natural equivalents. Moreover, the engineered silk proteins may be modified in order to gain a new function. The strategy of the hybrid proteins constructed on the DNA level combines the sequence of engineered silk, which is responsible for the biomaterial structure, with the sequence of polypeptide which allows functionalization of the silk biomaterial. The functional domains may comprise receptor binding sites, enzymes, metal or sugar binding sites and others. Currently, advanced research is being conducted, which on the one hand focuses on establishing the particular silk structure and understanding the process of silk thread formation in nature. On the other hand, there are attempts to improve methods of engineered spider silk protein production. Due to acquired knowledge and recent progress in synthetic protein technology, the engineered silk will turn into intelligent biomaterial of the future, while its industrial production scale will trigger a biotechnological revolution.

Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals.

PubMed

Fung, Ho Yee Joyce; Fu, Szu-Chin; Chook, Yuh Min

2017-03-10

Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs.

A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

PubMed

Brewer, J M; Wampler, J E

2001-03-14

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

Salt- and pH-Triggered Helix-Coil Transition of Ionic Polypeptides under Physiology Conditions.

PubMed

Yuan, Jingsong; Zhang, Yi; Sun, Yue; Cai, Zhicheng; Yang, Lijiang; Lu, Hua

2018-06-11

Controlling the helix-coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here, we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EG x )-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P(EG x DMA-Glu) ( x = 1, 2, and 3), show excellent aqueous solubility (>20 mg/mL) regardless of their charge states. Unlike poly-l-lysine that can form a helix only at pH above 10, P(EG x DMA-Glu) undergo a pH-dependent helix-coil switch with their transition points within the physiological range (pH ∼5.3-6.5). Meanwhile, P(EG x DMA-Glu) exhibit an unusual salt-induced helical conformation presumably owing to the unique properties of EG x linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides and represents a facile approach toward stimuli-responsive biopolymers for advanced biological applications.

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities.

PubMed

Suarez, M; Entenza, J M; Doerries, C; Meyer, E; Bourquin, L; Sutherland, J; Marison, I; Moreillon, P; Mermod, N

2003-01-05

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings. Copyright 2002 Wiley Periodicals, Inc.

Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

PubMed Central

Siroy, Axel; Molle, Virginie; Lemaître-Guillier, Christelle; Vallenet, David; Pestel-Caron, Martine; Cozzone, Alain J.; Jouenne, Thierry; Dé, Emmanuelle

2005-01-01

It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels. PMID:16304148

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 effects on peptide conformation. pi-orbital interactions at the molecular level were observed to be very sensitive to intermolecular distance and orientation of the chromophores attached to the alpha-helical peptide templates. When the methylstilbene or Oxa-PPV molecules were arranged on the same side of the helix with intermolecular spacing of 6A, the chromophores interacted strongly with each other forming excimers. Such interactions were absent when the molecules were arranged on the opposite side of the helix. These peptide-templated systems therefore offer enormous opportunities for the elucidation of complex photophysical phenomena that occur in relatively aggregated morphologies of conjugated species, but under dilute solution conditions in which the number of chromphores in the aggregate can be manipulated. Part 2. Synthesis and characterization of biocompatible polypeptide elastomer. Lately, the significance of mechanical forces and biological cues involved in tissue remodeling are highly valued; thus the capacity of a biomaterial to present a fitting mechanical and biological environment for optimal tissue generation has become a key parameter for biomaterial design. In addition to having suitable mechanical properties, materials used for these applications need to be biologically active, i.e. trigger dynamic interactions with cells and stimulate explicit cell and tissue responses. Thus, we have designed a resilin-based modular biomaterial incorporating both mechanically and biologically active domains to sense and aptly respond to the bio-mechanical demand or changes in their environment. The use of resilin-like polypeptides offers access to a class of hydrophilic elastomers with excellent resilience and high frequency responsiveness, which can be used for encapsulating hydrophilic drugs like proteins for drug delivery, and provides hydrophilic extracellular matrix mimicking cell adhesive and enzyme degradable substrate for tissue engineering. Hence, we have designed a multi-component elastomeric polypeptide scaffold containing a resilin mimetic flexible domain that governs the mechanical properties, a cell adhesion domain, a proteolytic degradation site and a growth factor binding domain for applications in tissue regeneration. The modular protein was synthesized in an E. coli host and the purified protein, as analyzed via CD and FTIR exhibited mainly unordered conformation with slight contribution from type II beta-turns. The crosslinked protein is highly elastic in nature and support cell attachment. There materials could be used for tissue repair where high frequency responsiveness and superior fatigue resistance are important, such as scaffolds for vocal fold regenerations and as cardiovascular patches.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella typhimurium.

PubMed

Bargieri, Daniel Y; Leite, Juliana A; Lopes, Stefanie C P; Sbrogio-Almeida, Maria Elisabete; Braga, Catarina J M; Ferreira, Luis C S; Soares, Irene S; Costa, Fabio T M; Rodrigues, Mauricio M

2010-04-01

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP)

PubMed Central

Watashi, Koichi; Sluder, Ann; Daito, Takuji; Matsunaga, Satoko; Ryo, Akihide; Nagamori, Shushi; Iwamoto, Masashi; Nakajima, Syo; Tsukuda, Senko; Borroto-Esoda, Katyna; Sugiyama, Masaya; Tanaka, Yasuhito; Kanai, Yoshikatsu; Kusuhara, Hiroyuki; Mizokami, Masashi; Wakita, Takaji

2014-01-01

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug-resistant viruses highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection-susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having a median inhibitory concentration <0.2 μM. Conclusion: This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. (Hepatology 2014;59:1726–1737) PMID:24375637

Peptide probes reveal a hydrophobic steric ratchet in the anthrax toxin protective antigen translocase

PubMed Central

Colby, Jennifer M.; Krantz, Bryan A.

2015-01-01

Anthrax toxin is a tripartite virulence factor produced by Bacillus anthracis during infection. Under acidic endosomal pH conditions, the toxin's protective antigen (PA) component forms a transmembrane channel in host cells. The PA channel then translocates its two enzyme components, lethal factor (LF) and edema factor (EF), into the host cytosol under the proton motive force (PMF). Protein translocation under a PMF is catalyzed by a series of nonspecific polypeptide binding sites, called clamps. A 10-residue guest/host peptide model system, KKKKKXXSXX, was used to functionally probe polypeptide-clamp interactions within wild-type PA channels. The guest residues were Thr, Ala, Leu, Phe, Tyr, and Trp. In steady-state translocation experiments, the channel blocked most tightly with peptides that had increasing amounts of nonpolar surface area. Cooperative peptide binding was observed in the Trp-containing peptide sequence but not the other tested sequences. Trp substitutions into a flexible, uncharged linker between LF amino-terminal domain and diphtheria toxin A chain expedited translocation. Therefore, peptide clamp sites in translocase channels can sense large steric features (like tryptophan) in peptides; and while these steric interactions may make a peptide translocate poorly, in the context of folded domains they can make the protein translocate more rapidly presumably via a hydrophobic steric ratchet mechanism. PMID:26363343

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, W.R.; Grossman, A.R.

One- and two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunological analyses were used to visualize differences in polypeptides synthesized by Symbiodinium sp. from the anemone Aiptasia pallida when grown in the cultured and endosymbiotic states (freshly isolated zooxanthellae). Surprisingly, a comparison of proteins in cultured and endosymbiotic Symbiodinium sp. revealed only four major polypeptides with similar isoelectric and molecular mass characteristics. Using monospecific antibodies, we demonstrated differences in specific proteins synthesized by the dinoflagellate in the two different growth states. The dimeric, 14 kDa form of the peripheral membrane peridinin-chlorophyll a binding protein predominates under endosymbiotic conditions, whereas the monomeric, 35more » kDa form predominates under the culture conditions used in this study. Antibodies to form II ribulose-1,5-bisphosphate carboxylase revealed 62 and 60 kDa forms of this protein in the alga grown as an endosymbiont and in culture, respectively. Differences in the integral membrane peridinin-chlorophyll a-c-binding proteins were also observed. These results demonstrate that there are major changes in the populations of proteins synthesized by Symbiodinium sp. in response to the conditions in hospite. Such changes may reflect a developmental switch that tailors the physiology of the alga to the conditions encountered in the endosymbiotic state. 77 refs., 6 figs.« less

Crystal structures of Hsp104 N-terminal domains from Saccharomyces cerevisiae and Candida albicans suggest the mechanism for the function of Hsp104 in dissolving prions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Peng; Li, Jingzhi; Weaver, Clarissa

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs fromSaccharomyces cerevisiae(ScHsp104NTD) andCandida albicans(CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around themore » putative peptide-binding groove mediate the monomer–monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.« less

Star Polymers Reduce Islet Amyloid Polypeptide Toxicity via Accelerated Amyloid Aggregation.

PubMed

Pilkington, Emily H; Lai, May; Ge, Xinwei; Stanley, William J; Wang, Bo; Wang, Miaoyi; Kakinen, Aleksandr; Sani, Marc-Antonie; Whittaker, Michael R; Gurzov, Esteban N; Ding, Feng; Quinn, John F; Davis, Thomas P; Ke, Pu Chun

2017-12-11

Protein aggregation into amyloid fibrils is a ubiquitous phenomenon across the spectrum of neurodegenerative disorders and type 2 diabetes. A common strategy against amyloidogenesis is to minimize the populations of toxic oligomers and protofibrils by inhibiting protein aggregation with small molecules or nanoparticles. However, melanin synthesis in nature is realized by accelerated protein fibrillation to circumvent accumulation of toxic intermediates. Accordingly, we designed and demonstrated the use of star-shaped poly(2-hydroxyethyl acrylate) (PHEA) nanostructures for promoting aggregation while ameliorating the toxicity of human islet amyloid polypeptide (IAPP), the peptide involved in glycemic control and the pathology of type 2 diabetes. The binding of PHEA elevated the β-sheet content in IAPP aggregates while rendering a new morphology of "stelliform" amyloids originating from the polymers. Atomistic molecular dynamics simulations revealed that the PHEA arms served as rodlike scaffolds for IAPP binding and subsequently accelerated IAPP aggregation by increased local peptide concentration. The tertiary structure of the star nanoparticles was found to be essential for driving the specific interactions required to impel the accelerated IAPP aggregation. This study sheds new light on the structure-toxicity relationship of IAPP and points to the potential of exploiting star polymers as a new class of therapeutic agents against amyloidogenesis.

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins.

PubMed

Deming, Timothy J

2017-03-15

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

Force field dependent solution properties of glycine oligomers

PubMed Central

Drake, Justin A.

2015-01-01

Molecular simulations can be used to study disordered polypeptide systems and to generate hypotheses on the underlying structural and thermodynamic mechanisms that govern their function. As the number of disordered protein systems investigated with simulations increase, it is important to understand how particular force fields affect the structural properties of disordered polypeptides in solution. To this end, we performed a comparative structural analysis of Gly3 and Gly10 in aqueous solution from all-atom, microsecond MD simulations using the CHARMM 27 (C27), CHARMM 36 (C36), and Amber ff12SB force fields. For each force field, Gly3 and Gly10 were simulated for at least 300 ns and 1 μs, respectively. Simulating oligoglycines of two different lengths allows us to evaluate how force field effects depend on polypeptide length. Using a variety of structural metrics (e.g. end-to-end distance, radius of gyration, dihedral angle distributions), we characterize the distribution of oligoglycine conformers for each force field and show that each sample conformation space differently, yielding considerably different structural tendencies of the same oligoglycine model in solution. Notably, we find that C36 samples more extended oligoglycine structures than both C27 and ff12SB. PMID:25952623

Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties.

PubMed

Thouless, M E; Wildy, P

1975-02-01

The kinetics of formation, the stability at 40 degrees C and the serological properties of thymidine kinase and deoxycytidine kinase activities induced by herpes simplex virus have been examined. The results are consistent with the hypothesis that both activities are carried on the same molecule-a deoxypyrimidine kinase. Mutants deficient in deoxypyrimidine kinase have been used to produce, by absorption of general antisera, deoxypyrimidine kinase-specific antisera. Using immunoprecipitation and SDS-polyacrylamide gel electrophoresis, only one size of polypeptide (mol. wt. 42400 plus or minus 200) has been found, constituting the type 2 enzyme. This is close to published values for the type i enzyme but co-electrophoresis demonstrated that the polypeptide of the type i enzyme was slightly bigger.

Design of monodisperse and well-defined polypeptide-based polyvalent inhibitors of anthrax toxin.

PubMed

Patke, Sanket; Boggara, Mohan; Maheshwari, Ronak; Srivastava, Sunit K; Arha, Manish; Douaisi, Marc; Martin, Jacob T; Harvey, Ian B; Brier, Matthew; Rosen, Tania; Mogridge, Jeremy; Kane, Ravi S

2014-07-28

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure-activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide-based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin-binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

PubMed

Stayton, M M; Black, M; Bedbrook, J; Dunsmuir, P

1986-12-22

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

The Mechanism and Function of Group II Chaperonins

DOE PAGES

Lopez, Tom; Dalton, Kevin; Frydman, Judith

2015-04-30

We report protein folding in the cell requires the assistance of enzymes collectively called chaperones. Among these, the chaperonins are 1-MDa ring-shaped oligomeric complexes that bind unfolded polypeptides and promote their folding within an isolated chamber in an ATP-dependent manner. Group II chaperonins, found in archaea and eukaryotes, contain a built-in lid that opens and closes over the central chamber. In eukaryotes, the chaperonin TRiC/CCT is hetero-oligomeric, consisting of two stacked rings of eight paralogous subunits each. TRiC facilitates folding of approximately 10% of the eukaryotic proteome, including many cytoskeletal components and cell cycle regulators. Folding of many cellular substratesmore » of TRiC cannot be assisted by any other chaperone. A complete structural and mechanistic understanding of this highly conserved and essential chaperonin remains elusive. However, recent work is beginning to shed light on key aspects of chaperonin function and how their unique properties underlie their contribution to maintaining cellular proteostasis.« less

Two-dimensional honeycomb network through sequence-controlled self-assembly of oligopeptides.

PubMed

Abb, Sabine; Harnau, Ludger; Gutzler, Rico; Rauschenbach, Stephan; Kern, Klaus

2016-01-12

The sequence of a peptide programs its self-assembly and hence the expression of specific properties through non-covalent interactions. A large variety of peptide nanostructures has been designed employing different aspects of these non-covalent interactions, such as dispersive interactions, hydrogen bonding or ionic interactions. Here we demonstrate the sequence-controlled fabrication of molecular nanostructures using peptides as bio-organic building blocks for two-dimensional (2D) self-assembly. Scanning tunnelling microscopy reveals changes from compact or linear assemblies (angiotensin I) to long-range ordered, chiral honeycomb networks (angiotensin II) as a result of removal of steric hindrance by sequence modification. Guided by our observations, molecular dynamic simulations yield atomistic models for the elucidation of interpeptide-binding motifs. This new approach to 2D self-assembly on surfaces grants insight at the atomic level that will enable the use of oligo- and polypeptides as large, multi-functional bio-organic building blocks, and opens a new route towards rationally designed, bio-inspired surfaces.

Statistical thermodynamics of protein folding: Comparison of a mean-field theory with Monte Carlo simulations

NASA Astrophysics Data System (ADS)

Hao, Ming-Hong; Scheraga, Harold A.

1995-01-01

A comparative study of protein folding with an analytical theory and computer simulations, respectively, is reported. The theory is based on an improved mean-field formalism which, in addition to the usual mean-field approximations, takes into account the distributions of energies in the subsets of conformational states. Sequence-specific properties of proteins are parametrized in the theory by two sets of variables, one for the energetics of mean-field interactions and one for the distribution of energies. Simulations are carried out on model polypeptides with different sequences, with different chain lengths, and with different interaction potentials, ranging from strong biases towards certain local chain states (bond angles and torsional angles) to complete absence of local conformational preferences. Theoretical analysis of the simulation results for the model polypeptides reveals three different types of behavior in the folding transition from the statistical coiled state to the compact globular state; these include a cooperative two-state transition, a continuous folding, and a glasslike transition. It is found that, with the fitted theoretical parameters which are specific for each polypeptide under a different potential, the mean-field theory can describe the thermodynamic properties and folding behavior of the different polypeptides accurately. By comparing the theoretical descriptions with simulation results, we verify the basic assumptions of the theory and, thereby, obtain new insights about the folding transitions of proteins. It is found that the cooperativity of the first-order folding transition of the model polypeptides is determined mainly by long-range interactions, in particular the dipolar orientation; the local interactions (e.g., bond-angle and torsion-angle potentials) have only marginal effect on the cooperative characteristic of the folding, but have a large impact on the difference in energy between the folded lowest-energy structure and the unfolded conformations of a protein.

Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting Polypeptide

PubMed Central

Yan, Huan; Peng, Bo; Liu, Yang; Xu, Guangwei; He, Wenhui; Ren, Bijie; Jing, Zhiyi; Sui, Jianhua

2014-01-01

ABSTRACT The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs. IMPORTANCE Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection. PMID:24390325

A Developmental Study of Photosystem I Peripheral Chlorophyll Proteins 1

PubMed Central

Mullet, John E.; Burke, John J.; Arntzen, Charles J.

1980-01-01

An isolated “native” photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae (≃45 Chl/P700) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association. Chloroplasts were isolated from a Chl b-less barley mutant and from partially-developed cucumber cotyledons (greened under intermittent illumination [ImL] chloroplasts) and were compared to control chloroplasts isolated from wild-type barley and mature cucumber. Both the mutant and ImL chloroplasts exhibited a long wavelength fluorescence maximum at 724 nanometers at 77 K as compared to 735 to 738 nanometers emission maximum in the respective controls. Both the mutant and ImL chloroplasts were deficient in polypeptides of 21,500 to 24,500 daltons which were present in control membranes and in PSI fractions isolated from control membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range paralleled the appearance of PSI Chl species fluorescing at long wavelength (≃735 nm). The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 Chl/P700 (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus ≃ 45 Chl/P700. The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts. Images PMID:16661289

Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

PubMed Central

Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

1997-01-01

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a K d of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

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 pH values. Variations in fiber morphology, elemental composition and stability have been studied by microscopy and energy-dispersive X-ray spectroscopy (EDX), following the treatment of samples at different pH values in the 2-12 range. Fiber stability has been interpreted with reference to the pH dependence of the UV absorbance and fluorescence of PLEY chains in solution. The data show that fiber stability is crucially dependent on the extent of side chain ionization, even after crosslinking. Self-organization kinetics of electrospun PLO and PLEY fibers during solvent annealing has been studied. After being crosslinked in situ , fibers were annealed in water at 22 °C. Analysis by Fourier transform infrared spectroscopy (FTIR) has revealed that annealing involved fiber restructuring with an overall time constant of 29 min for PLO and 63 min for PLEY, and that changes in the distribution of polymer conformations occurred during the first 13 min of annealing. There was a substantial decrease in the amount of Na+ bound to PLEY fibers during annealing. Kinetic modeling has indicated that two parallel pathways better account for the annealing trajectory than a single pathway with multiple transition states. Taken together, the results will advance the rational design of polypeptides for peptide-based materials, especially materials prepared by electrospinning. It is believed that this research will increase basic knowledge of polymer electrospinning and advance the development of electrospun materials, especially in medicine and biotechnology. The study has yielded two advances on previous work in the area: avoidance of an animal source of peptides and avoidance of inorganic solvent. The present results thus advance the growing field of peptide-based materials. Non-woven electrospun fiber mats made of polypeptides are increasingly considered attractive for basic research and technology development in biotechnology, medicine and other areas. (Abstract shortened by UMI.)

Clostridium Perfringens Enterotoxin (CPE) and CPE-Binding Domain (c-CPE) for the Detection and Treatment of Gynecologic Cancers

PubMed Central

Black, Jonathan D.; Lopez, Salvatore; Cocco, Emiliano; Schwab, Carlton L.; English, Diana P.; Santin, Alessandro D.

2015-01-01

Clostridium perfringens enterotoxin (CPE) is a three-domain polypeptide, which binds to Claudin-3 and Claudin-4 with high affinity. Because these receptors are highly differentially expressed in many human tumors, claudin-3 and claudin-4 may provide an efficient molecular tool to specifically identify and target biologically aggressive human cancer cells for CPE-specific binding and cytolysis. In this review we will discuss these surface proteins as targets for the detection and treatment of chemotherapy-resistant gynecologic malignancies overexpressing claudin-3 and -4 using CPE-based theranostic agents. We will also discuss the use of fluorescent c-CPE peptide in the operative setting for real time detection of micro-metastatic tumors during surgery and review the potential role of CPE in other medical applications. PMID:25835384

In vivo disruption of T cell development by expression of a dominant-negative polypeptide designed to abolish the SLP-76/Gads interaction.

PubMed

Jordan, Martha S; Maltzman, Jonathan S; Kliche, Stefanie; Shabason, Jacob; Smith, Jennifer E; Obstfeld, Amrom; Schraven, Burkhart; Koretzky, Gary A

2007-10-01

Multi-molecular complexes nucleated by adaptor proteins play a central role in signal transduction. In T cells, one central axis consists of the assembly of several signaling proteins linked together by the adaptors linker of activated T cells (LAT), Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads). Each of these adaptors has been shown to be important for normal T cell development, and their proper sub-cellular localization is critical for optimal function in cell lines. We previously demonstrated in Jurkat T cells and a rat basophilic leukemic cell line that expression of a 50-amino acid polypeptide identical to the site on SLP-76 that binds to Gads blocks proper localization of SLP-76 and SLP-76-dependent signaling events. Here we extend these studies to investigate the ability of this polypeptide to inhibit TCR-induced integrin activity in Jurkat cells and to inhibit in vivo thymocyte development and primary T cell function. These data provide evidence for the in vivo function of a dominant-negative peptide based upon the biology of SLP-76 action and suggest the possibility of therapeutic potential of targeting the SLP-76/Gads interaction.

Active Immunization Against hIAPP Oligomers Ameliorates the Diabetes- Associated Phenotype in a Transgenic Mice Model.

PubMed

Bram, Yaron; Peled, Sivan; Brahmachari, Sayanti; Harlev, Michael; Gazit, Ehud

2017-10-25

Type 2 diabetes is characterized by insulin tolerance in target cells followed by a reduction of pancreatic β-cell mass. Islet amyloid polypeptide oligomeric assemblies were shown to contribute to β-cell apoptosis by forming discrete pores that destabilize the cellular membrane. We previously characterized α-helical cytotoxic islet amyloid polypeptide oligomers which interact with cell membranes, following a complete internalization that leads to cellular apoptosis. Moreover, antibodies which bind the oligomers and neutralize the cytotoxicity were exclusively identified in the serum of type 2 diabetes patients. Here, we examined the usage of the newly characterized oligomers as an active immunization agent targeting amyloid self- assembly in a diabetes-associated phenotype transgenic mice model. Immunized transgenic mice showed an increase in hIAPP-antibody serum titer as well as improvement in diabetes-associated parameters. Lower fasting blood glucose levels, higher insulin, and lower islet amyloid polypeptide accumulation were observed. Furthermore, antibodies derived from the immunized mice reduced hIAPP oligomers cytotoxicity towards β-cells in a dose-dependent manner. This study highlights the significance of targeting the early amyloid self-assembly events for potential disease management. Furthermore, it demonstrates that α-helical oligomers conformers are valid epitope for the development of future immunization therapy.

Theoretical and computational validation of the Kuhn barrier friction mechanism in unfolded proteins.

PubMed

Avdoshenko, Stanislav M; Das, Atanu; Satija, Rohit; Papoian, Garegin A; Makarov, Dmitrii E

2017-03-21

A long time ago, Kuhn predicted that long polymers should approach a limit where their global motion is controlled by solvent friction alone, with ruggedness of their energy landscapes having no consequences for their dynamics. In contrast, internal friction effects are important for polymers of modest length. Internal friction in proteins, in particular, affects how fast they fold or find their binding targets and, as such, has attracted much recent attention. Here we explore the molecular origins of internal friction in unfolded proteins using atomistic simulations, coarse-grained models and analytic theory. We show that the characteristic internal friction timescale is directly proportional to the timescale of hindered dihedral rotations within the polypeptide chain, with a proportionality coefficient b that is independent of the chain length. Such chain length independence of b provides experimentally testable evidence that internal friction arises from concerted, crankshaft-like dihedral rearrangements. In accord with phenomenological models of internal friction, we find the global reconfiguration timescale of a polypeptide to be the sum of solvent friction and internal friction timescales. At the same time, the time evolution of inter-monomer distances within polypeptides deviates both from the predictions of those models and from a simple, one-dimensional diffusion model.

Conformational energy calculations on polypeptides and proteins: use of a statistical mechanical procedure for evaluating structure and properties.

PubMed

Scheraga, H A; Paine, G H

1986-01-01

We are using a variety of theoretical and computational techniques to study protein structure, protein folding, and higher-order structures. Our earlier work involved treatments of liquid water and aqueous solutions of nonpolar and polar solutes, computations of the stabilities of the fundamental structures of proteins and their packing arrangements, conformations of small cyclic and open-chain peptides, structures of fibrous proteins (collagen), structures of homologous globular proteins, introduction of special procedures as constraints during energy minimization of globular proteins, and structures of enzyme-substrate complexes. Recently, we presented a new methodology for predicting polypeptide structure (described here); the method is based on the calculation of the probable and average conformation of a polypeptide chain by the application of equilibrium statistical mechanics in conjunction with an adaptive, importance sampling Monte Carlo algorithm. As a test, it was applied to Met-enkephalin.

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.

Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines.

PubMed

Despanie, Jordan; Dhandhukia, Jugal P; Hamm-Alvarez, Sarah F; MacKay, J Andrew

2016-10-28

Elastin-like polypeptides (ELPs) constitute a genetically engineered class of 'protein polymers' derived from human tropoelastin. They exhibit a reversible phase separation whereby samples remain soluble below a transition temperature (T t ) but form amorphous coacervates above T t . Their phase behavior has many possible applications in purification, sensing, activation, and nanoassembly. As humanized polypeptides, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators. This review aims to offer the reader a catalogue of ELPs, their various applications, and potential for commercialization across a broad spectrum of fields. Copyright © 2015. Published by Elsevier B.V.

Detecting and Targeting Oncogenic Myc in Breast Cancer

DTIC Science & Technology

2006-06-01

expression in lung tumor samples. Real-time quantitative PCR amplification was conducted using the SYBR Green assay in the ABI PRISM 7900-HT (Applied...Methylation-sensitive se- quence-specific DNA binding by the c-Myc basic region. Science 1991;251:186–9. 37. Perini G, Diolaiti D, Porro A, et al. In...using nuclear magnetic resonance and circular dichroism. We show that several Myc NTD polypeptides are largely disordered in solution, which is

Calculation of relative free energies for ligand-protein binding, solvation, and conformational transitions using the GROMOS software.

PubMed

Riniker, Sereina; Christ, Clara D; Hansen, Halvor S; Hünenberger, Philippe H; Oostenbrink, Chris; Steiner, Denise; van Gunsteren, Wilfred F

2011-11-24

The calculation of the relative free energies of ligand-protein binding, of solvation for different compounds, and of different conformational states of a polypeptide is of considerable interest in the design or selection of potential enzyme inhibitors. Since such processes in aqueous solution generally comprise energetic and entropic contributions from many molecular configurations, adequate sampling of the relevant parts of configurational space is required and can be achieved through molecular dynamics simulations. Various techniques to obtain converged ensemble averages and their implementation in the GROMOS software for biomolecular simulation are discussed, and examples of their application to biomolecules in aqueous solution are given. © 2011 American Chemical Society

An intracellular protein that binds amyloid-β peptide and mediates neurotoxicity in Alzheimer's disease

NASA Astrophysics Data System (ADS)

Du Yan, Shi; Fu, Jin; Soto, Claudio; Chen, Xi; Zhu, Huaijie; Al-Mohanna, Futwan; Collison, Kate; Zhu, Aiping; Stern, Eric; Saido, Takaomi; Tohyama, Masaya; Ogawa, Satoshi; Roher, Alex; Stern, David

1997-10-01

Amyloid-β is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimer's disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimer's disease. ERAB immunoprecipitates with amyloid-β, and when cell cultures are exposed to amyloid-β, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-β on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-β, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimer's disease.

Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN

PubMed Central

Elf, Shannon; Abdelfattah, Nouran S.; Baral, April J.; Beeson, Danielle; Rivera, Jeanne F.; Ko, Amy; Florescu, Natalie; Birrane, Gabriel; Chen, Edwin

2018-01-01

Mutations in calreticulin (CALR) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms. PMID:29288169

Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN.

PubMed

Elf, Shannon; Abdelfattah, Nouran S; Baral, April J; Beeson, Danielle; Rivera, Jeanne F; Ko, Amy; Florescu, Natalie; Birrane, Gabriel; Chen, Edwin; Mullally, Ann

2018-02-15

Mutations in calreticulin ( CALR ) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms. © 2018 by The American Society of Hematology.

Analysis of a Viral Agent Isolated from Multiple Sclerosis Brain Tissue: Characterization as a Parainfluenzavirus Type 1

PubMed Central

Lewandowski, L. J.; Lief, F. S.; Verini, M. A.; Pienkowski, M. M.; ter Meulen, V.; Koprowski, H.

1974-01-01

A virus originally isolated from cell cultures obtained by lysolecithin-induced fusion of human multiple sclerosis brain cells with CV-1 cells has been analyzed for its antigenic, RNA, and polypeptide compositions, and for selective biological properties. Our findings establish that this isolate, designated 6/94 virus, contains a 50S RNA genome and is, as yet, indistinguishable from Sendai virus in its antigenic and total polypeptide compositions. Despite these similarities, the 6/94 and Sendai viruses differ in certain phenotypic properties. 6/94 virus is markedly less cytocidal for chick fibroblasts, especially at 37 C and, after β-propiolactone inactivation, it possesses a greater capacity for cell fusion and a lower toxicity than does comparably treated Sendai virus. In addition, 6/94 virus shows greater hemolytic activity. Images PMID:4363249

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

PubMed

Keeley, Fred W; Bellingham, Catherine M; Woodhouse, Kimberley A

2002-02-28

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

How Lipid Membranes Affect Pore Forming Toxin Activity.

PubMed

Rojko, Nejc; Anderluh, Gregor

2015-12-15

Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally, events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein-lipid membrane interactions in greater detail.

Interaction of fluvastatin with the liver-specific Na+ -dependent taurocholate cotransporting polypeptide (NTCP).

PubMed

Greupink, Rick; Dillen, Lieve; Monshouwer, Mario; Huisman, Maarten T; Russel, Frans G M

2011-11-20

It has been reported that polymorphisms in the organic anion transporting polypeptide 1B1 (OATP1B1, SLCO1B1) result in decreased hepatic uptake of simvastatin carboxy acid, the active metabolite of simvastatin. This is not the case for fluvastatin and it has been hypothesized that for this drug other hepatic uptake pathways exist. Here, we studied whether Na(+)-dependent taurocholate co-transporting polypeptide (NTCP, SLC10A1) can be an alternative hepatic uptake route for fluvastatin. Chinese Hamster Ovary cells transfected with human NTCP (CHO-NTCP) were used to investigate the inhibitory effect of fluvastatin and other statins on [(3)H]-taurocholic acid uptake ([(3)H]-TCA). Statin uptake by CHO-NTCP and cryopreserved human hepatocytes was assessed via LC-MS/MS. Fluvastatin appeared to be a potent and competitive inhibitor of [(3)H]-TCA uptake (IC(50) of 40μM), pointing to an interaction at the level of the bile acid binding pocket of NTCP. The inhibitory action of other statins was also studied, which revealed that statin inhibitory potency increased with molecular descriptors of lipophilicity: calculated logP (r(2)=0.82, p=0.034), logD(7.4) (r(2)=0.77, p=0.0001). Studies in CHO-NTCP cells showed that fluvastatin was indeed an NTCP substrate (K(m) 250±30μM, V(max) 1340±50ng/mg total cell protein/min). However, subsequent studies revealed that at clinically relevant plasma concentrations, NTCP contributed minimally to overall accumulation in human hepatocytes. In conclusion, fluvastatin interacts with NTCP at the level of the bile acid binding pocket and is an NTCP substrate. However, under normal conditions, NTCP-mediated uptake of this drug seems not to be a significant hepatocellular uptake pathway. Copyright © 2011 Elsevier B.V. All rights reserved.

The Design and Delivery of a Thermally Responsive Peptide to Inhibit S100B Mediated Neurodegeneration

PubMed Central

Hearst, Scoty M; Walker, Leslie R; Shao, Qingmei; Lopez, Mariper; Raucher, Drazen; Vig, Parminder J S

2011-01-01

S100B, a glial secreted protein is believed to play a major role in neurodegeneration in Alzheimer's disease, Down syndrome, traumatic brain injury and spinocerebellar ataxia type 1 (SCA1). SCA1 is a trinucleotide repeat disorder in which the expanded polyglutamine mutation in the protein ataxin-1 primarily targets Purkinje cells (PCs) of the cerebellum. Currently, the exact mechanism of S100B mediated PC damage in SCA1 is not clear. However, here we show that S100B may act via the activation of the RAGE signaling pathway resulting in oxidative stress mediated injury to mutant ataxin-1 expressing neurons. To combat S100B mediated neurodegeneration, we have designed a selective thermally responsive S100B inhibitory peptide, Synb1-ELP-TRTK. Our therapeutic polypeptide was developed using three key elements: (1) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (2) the TRTK12 peptide, a known S100B inhibitory peptide and (3) a cell penetrating peptide, Synb1, to enhance intracellular delivery. Binding studies revealed that our peptide, Synb1-ELP-TRTK, interacts with its molecular target S100B and maintains a high S100B binding affinity as comparable with the TRTK12 peptide alone. In addition, in vitro studies revealed that Synb1-ELP-TRTK treatment reduces S100B uptake in SHSY5Y cells. Furthermore, the Synb1-ELP-TRTK peptide decreased S100B induced oxidative damage to mutant ataxin-1 expressing neurons. To test the delivery capabilities of ELP based therapeutic peptides to the cerebellum; we treated mice with fluorescently labeled Synb1-ELP and observed that thermal targeting enhanced peptide delivery to the cerebellum. Here, we have laid the framework for thermal based therapeutic targeting to regions of the brain, particularly the cerebellum. Overall, our data suggests that thermal targeting of ELP based therapeutic peptides to the cerebellum is a novel treatment strategy for cerebellar neurodegenerative disorders. PMID:21958864

Reversible Opening of the Blood-Brain Barrier by Anti-Bacterial Antibodies

NASA Astrophysics Data System (ADS)

Tuomanen, Elaine I.; Prasad, Sudha M.; George, Jonathan S.; Hoepelman, Andy I. M.; Ibsen, Per; Heron, Iver; Starzyk, Ruth M.

1993-08-01

The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells. Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3. We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability. Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits. Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18. In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma.

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.

PubMed

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.

Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

PubMed

Laman, A G; Lathe, R; Savinov, G V; Shepelyakovskaya, A O; Boziev, Kh M; Baidakova, L K; Chulin, A N; Brovko, F A; Svirshchevskaya, E V; Kotelevtsev, Y; Eliseeva, I A; Guryanov, S G; Lyabin, D N; Ovchinnikov, L P; Ivanov, V T

2015-07-08

The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

PubMed Central

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

Antibody neutralization of retargeted measles viruses.

PubMed

Lech, Patrycja J; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J; Nara, Peter L; Russell, Stephen J

2014-04-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

NASA Astrophysics Data System (ADS)

Miles, Jeff; Formosa, Tim

1992-02-01

We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages and Conjugative Elements

PubMed Central

Iyer, Lakshminarayan M.; Burroughs, A. Maxwell; Anand, Swadha; de Souza, Robson F.

2017-01-01

ABSTRACT Intense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular “weaponry” deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance. IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems. PMID:28559295

Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

PubMed

König, Alexander; Glebe, Dieter

2017-01-01

To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes.

PubMed

König, Alexander; Döring, Barbara; Mohr, Christina; Geipel, Andreas; Geyer, Joachim; Glebe, Dieter

2014-10-01

The human liver bile acid transporter Na(+)/taurocholate cotransporting polypeptide (NTCP) has recently been identified as liver-specific receptor for infection of hepatitis B virus (HBV), which attaches via the myristoylated preS1 (myr-preS1) peptide domain of its large surface protein to NTCP. Since binding of the myr-preS1 peptide to NTCP is an initiating step of HBV infection, we investigated if this process interferes with the physiological bile acid transport function of NTCP. HBV infection, myr-preS1 peptide binding, and bile acid transport assays were performed with primary Tupaia belangeri (PTH) and human (PHH) hepatocytes as well as NTCP-transfected human hepatoma HepG2 cells allowing regulated NTCP expression, in the presence of various bile acids, ezetimibe, and myr-preS1 peptides. The myr-preS1 peptide of HBV inhibited bile acid transport in PTH and PHH as well as in NTCP-expressing HEK293 and HepG2 cells. Inversely, HBV infection of PTH, PHH, and NTCP-transfected HepG2 cells was inhibited in a concentration-dependent manner by taurine and glycine conjugates of cholic acid and ursodeoxycholic acid as well as by ezetimibe. In NTCP-HepG2 cells and PTH, NTCP expression, NTCP transport function, myr-preS1 peptide binding, and HBV infection followed comparable kinetics. Myr-preS1 virus binding to NTCP, necessary for productive HBV infection, interferes with the physiological bile acid transport function of NTCP. Therefore, HBV infection via NTCP may be lockable by NTCP substrates and NTCP-inhibiting drugs. This opens a completely new way for an efficient management of HBV infection by the use of NTCP-directed drugs. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Characterisation of the hepatitis B virus cross-species transmission pattern via Na+/taurocholate co-transporting polypeptides from 11 New World and Old World primate species.

PubMed

Müller, Simon F; König, Alexander; Döring, Barbara; Glebe, Dieter; Geyer, Joachim

2018-01-01

The hepatic Na+/taurocholate co-transporting polypeptide (NTCP in man, Ntcp in animals) is the high-affinity receptor for the hepatitis B (HBV) and hepatitis D (HDV) viruses. Species barriers for human HBV/HDV within the order Primates were previously attributed to Ntcp sequence variations that disable virus-receptor interaction. However, only a limited number of primate Ntcps have been analysed so far. In the present study, a total of 11 Ntcps from apes, Old and New World monkeys were cloned and expressed in vitro to characterise their interaction with HBV and HDV. All Ntcps showed intact bile salt transport. Human NTCP as well as the Ntcps from the great apes chimpanzee and orangutan showed transport-competing binding of HBV derived myr-preS1-peptides. In contrast, all six Ntcps from the group of Old World monkeys were insensitive to HBV myr-preS1-peptide binding and HBV/HDV infection. This is basically predetermined by the amino acid arginine at position 158 of all studied Old World monkey Ntcps. An exchange from arginine to glycine (as present in humans and great apes) at this position (R158G) alone was sufficient to achieve full transport-competing HBV myr-preS1-peptide binding and susceptibility for HBV/HDV infection. New World monkey Ntcps showed higher sequence heterogeneity, but in two cases with 158G showed transport-competing HBV myr-preS1-peptide binding, and in one case (Saimiri sciureus) even susceptibility for HBV/HDV infection. In conclusion, amino acid position 158 of NTCP/Ntcp is sufficient to discriminate between the HBV/HDV susceptible group of humans and great apes (158G) and the non-susceptible group of Old World monkeys (158R). In the case of the phylogenetically more distant New World monkey Ntcps amino acid 158 plays a significant, but not exclusive role.

A conserved START domain coenzyme Q-binding polypeptide is required for efficient Q biosynthesis, respiratory electron transport, and antioxidant function in Saccharomyces cerevisiae.

PubMed

Allan, Christopher M; Hill, Shauna; Morvaridi, Susan; Saiki, Ryoichi; Johnson, Jarrett S; Liau, Wei-Siang; Hirano, Kathleen; Kawashima, Tadashi; Ji, Ziming; Loo, Joseph A; Shepherd, Jennifer N; Clarke, Catherine F

2013-04-01

Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain. Copyright © 2012 Elsevier B.V. All rights reserved.

Targeting allosteric disulphide bonds in cancer.

PubMed

Hogg, Philip J

2013-06-01

Protein action in nature is generally controlled by the amount of protein produced and by chemical modification of the protein, and both are often perturbed in cancer. The amino acid side chains and the peptide and disulphide bonds that bind the polypeptide backbone can be post-translationally modified. Post-translational cleavage or the formation of disulphide bonds are now being identified in cancer-related proteins and it is timely to consider how these allosteric bonds could be targeted for new therapies.

J chain in the nurse shark: implications for function in a lower vertebrate.

PubMed

Hohman, Valerie S; Stewart, Sue E; Rumfelt, Lynn L; Greenberg, Andrew S; Avila, David W; Flajnik, Martin F; Steiner, Lisa A

2003-06-15

J chain is a small polypeptide covalently attached to polymeric IgA and IgM. In humans and mice, it plays a role in binding Ig to the polymeric Ig receptor for transport into secretions. The putative orthologue of mammalian J chain has been identified in the nurse shark by sequence analysis of cDNA and the polypeptide isolated from IgM. Conservation with J chains from other species is relatively poor, especially in the carboxyl-terminal portion, and, unlike other J chains, the shark protein is not acidic. The only highly conserved segment in all known J chains is a block of residues surrounding an N-linked glycosylation site. Of the eight half-cystine residues that are conserved in mammalian J chains, three are lacking in the nurse shark, including two in the carboxyl-terminal segment that have been reported to be required for binding of human J chain-containing IgA to secretory component. Taken together with these data, the relative abundance of J chain transcripts in the spleen and their absence in the spiral valve (intestine) suggest that J chain in nurse sharks may not have a role in Ig secretion. Analysis of J chain sequences in diverse species is in agreement with accepted phylogenetic relationships, with the exception of the earthworm, suggesting that the reported presence of J chain in invertebrates should be reassessed.

Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).

PubMed

Watashi, Koichi; Sluder, Ann; Daito, Takuji; Matsunaga, Satoko; Ryo, Akihide; Nagamori, Shushi; Iwamoto, Masashi; Nakajima, Syo; Tsukuda, Senko; Borroto-Esoda, Katyna; Sugiyama, Masaya; Tanaka, Yasuhito; Kanai, Yoshikatsu; Kusuhara, Hiroyuki; Mizokami, Masashi; Wakita, Takaji

2014-05-01

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug-resistant viruses highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection-susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having a median inhibitory concentration <0.2 μM. This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. Copyright © 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases.

Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process.

PubMed Central

Bartsevich, V V; Pakrasi, H B

1995-01-01

During photosynthesis, the photosystem II (PSII) pigment-protein complex catalyzes oxygen evolution, a reaction in which a four-manganese ensemble plays a crucial role. Using a newly developed selection scheme, we have isolated BP13, a random photosynthesis-deficient mutant strain of the cyanobacterium, Synechocystis 6803. This mutant grew slowly under photoautotrophic conditions, and had a low oxygen evolution activity. Biochemical analysis revealed that the lesion in this mutant strain had specifically affected the Mn ensemble in PSII. Interestingly, incubation of BP13 cells with micromolar levels of added Mn induced rapid recovery of oxygen evolution activity. The mutant could be complemented with a fragment of wild-type chromosomal DNA containing three closely linked genes, mntA, mntB and mntC. These gene products showed significant sequence similarities with polypeptide components of bacterial permeases that are members of the 'ABC (ATP binding cassette) superfamily' of transporter proteins. We determined that in the BP13 strain, a single nucleotide change had resulted in the replacement of an alanine by an aspartic acid residue in MntA, a soluble protein containing ATP binding motifs. These results suggest that the mntCAB gene cluster encodes polypeptide components of a Mn transporter, the first such protein complex identified in any organism. PMID:7743991

Peptide Probes Reveal a Hydrophobic Steric Ratchet in the Anthrax Toxin Protective Antigen Translocase.

PubMed

Colby, Jennifer M; Krantz, Bryan A

2015-11-06

Anthrax toxin is a tripartite virulence factor produced by Bacillus anthracis during infection. Under acidic endosomal pH conditions, the toxin's protective antigen (PA) component forms a transmembrane channel in host cells. The PA channel then translocates its two enzyme components, lethal factor and edema factor, into the host cytosol under the proton motive force. Protein translocation under a proton motive force is catalyzed by a series of nonspecific polypeptide binding sites, called clamps. A 10-residue guest/host peptide model system, KKKKKXXSXX, was used to functionally probe polypeptide-clamp interactions within wild-type PA channels. The guest residues were Thr, Ala, Leu, Phe, Tyr, and Trp. In steady-state translocation experiments, the channel blocked most tightly with peptides that had increasing amounts of nonpolar surface area. Cooperative peptide binding was observed in the Trp-containing peptide sequence but not the other tested sequences. Trp substitutions into a flexible, uncharged linker between the lethal factor amino-terminal domain and diphtheria toxin A chain expedited translocation. Therefore, peptide-clamp sites in translocase channels can sense large steric features (like tryptophan) in peptides, and while these steric interactions may make a peptide translocate poorly, in the context of folded domains, they can make the protein translocate more rapidly presumably via a hydrophobic steric ratchet mechanism. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Structure of the Poliovirus 135S Cell Entry Intermediate at 10-Angstrom Resolution Reveals the Location of an Externalized Polypeptide That Binds to Membranes†

PubMed Central

Bubeck, Doryen; Filman, David J.; Cheng, Naiqian; Steven, Alasdair C.; Hogle, James M.; Belnap, David M.

2005-01-01

Poliovirus provides a well-characterized system for understanding how nonenveloped viruses enter and infect cells. Upon binding its receptor, poliovirus undergoes an irreversible conformational change to the 135S cell entry intermediate. This transition involves shifts of the capsid protein β barrels, accompanied by the externalization of VP4 and the N terminus of VP1. Both polypeptides associate with membranes and are postulated to facilitate entry by forming a translocation pore for the viral RNA. We have calculated cryo-electron microscopic reconstructions of 135S particles that permit accurate placement of the β barrels, loops, and terminal extensions of the capsid proteins. The reconstructions and resulting models indicate that each N terminus of VP1 exits the capsid though an opening in the interface between VP1 and VP3 at the base of the canyon that surrounds the fivefold axis. Comparison with reconstructions of 135S particles in which the first 31 residues of VP1 were proteolytically removed revealed that the externalized N terminus is located near the tips of propeller-like features surrounding the threefold axes rather than at the fivefold axes, as had been proposed in previous models. These observations have forced a reexamination of current models for the role of the 135S particle in transmembrane pore formation and suggest testable alternatives. PMID:15919927

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

PubMed Central

Dayal, R; Hurlimann, J; Suard, Y M; Kraehenbuhl, J P

1982-01-01

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6177316

Binding of alpha-fetoprotein by immobilized monoclonal antibodies during episodes of zero-gravity obtained by parabolic flight

NASA Technical Reports Server (NTRS)

Spooner, Brian S.; Guikema, James A.; Barnes, Grady

1990-01-01

Alpha-fetoprotein (AFP), a single-chain polypeptide which is synthesized by the liver and yolk sac of the human fetus, provided a model ligand for assessing the effects of microgravity on ligand binding to surface-immobilized model receptor molecules. Monoclonal antibodies, used as receptors for AFP, were immobilized by covalent attachment to latex microparticles. Zero gravity environment was obtained by parabolic flight aboard NASA 930, a modified KC-135 aircraft. Buring the onset of an episode of zero gravity, ligand and receptor were mixed. Timed incubation (20 s) was terminated by centrifugation, the supernatant removed, and microparticies were assessed for bound AFP by immunochemical methods. The extent of binding was not influenced by microgravity, when compared with 1-G controls, which suggests that aberrant cellular activities observed in microgravity are not the simple expression of altered macromolecular interactions.

The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes

PubMed Central

Feng, Qin; Zhang, Yi

2001-01-01

Histone deacetylation plays an important role in methylated DNA silencing. Recent studies indicated that the methyl-CpG-binding protein, MBD2, is a component of the MeCP1 histone deacetylase complex. Interestingly, MBD2 is able to recruit the nucleosome remodeling and histone deacetylase, NuRD, to methylated DNA in vitro. To understand the relationship between the MeCP1 complex and the NuRD complex, we purified the MeCP1 complex to homogeneity and found that it contains 10 major polypeptides including MBD2 and all of the known NuRD components. Functional analysis of the purified MeCP1 complex revealed that it preferentially binds, remodels, and deacetylates methylated nucleosomes. Thus, our study defines the MeCP1 complex, and provides biochemical evidence linking nucleosome remodeling and histone deacetylation to methylated gene silencing. PMID:11297506

The binding of sodium dodecyl sulphate to various proteins

PubMed Central

Pitt-Rivers, Rosalind; Impiombato, F. S. Ambesi

1968-01-01

1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90–100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70–100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S·S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S·S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein–sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000–41000) irrespective of the molecular weight of the protein to which they were attached. PMID:4177067

Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.

PubMed

Treves, S; Feriotto, G; Moccagatta, L; Gambari, R; Zorzato, F

2000-12-15

Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions.

PubMed

Silver, Simon; Phung, Le T

2005-12-01

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups.

PubMed Central

Hickling, T. P.; Malhotra, R.; Sim, R. B.

1998-01-01

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events. Images Fig. 4 FIG. 6 Fig. 7 Fig. 8 PMID:9606179

Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5'-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins.

PubMed Central

Rancour, D M; Menon, A K

1998-01-01

Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide-sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[alpha-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[alpha-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[alpha-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed. PMID:9677326

Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

PubMed Central

Caldwell, Shane J.

2012-01-01

Aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5′-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (RG) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage. PMID:22290965

An Aß concatemer with altered aggregation propensities.

PubMed

Giehm, L; Dal Degan, F; Fraser, P; Klysner, S; Otzen, Daniel E

2010-10-01

We present an analysis of the conformational and aggregative properties of an Aß concatemer (Con-Alz) of interest for vaccine development against Alzheimer's disease. Con-Alz consists of 3 copies of the 43 residues of the Aß peptide separated by the P2 and P30 T-cell epitopes from the tetanus toxin. Even in the presence of high concentrations of denaturants or fluorinated alcohols, Con-Alz has a very high propensity to form aggregates which slowly coalesce over time with changes in secondary, tertiary and quaternary structure. Only micellar concentrations of SDS were able to inhibit aggregation. The increase in the ability to bind the fibril-binding dye ThT increases without lag time, which is characteristic of relatively amorphous aggregates. Confirming this, electron microscopy reveals that Con-Alz adopts a morphology resembling truncated protofibrils after prolonged incubation, but it is unable to assemble into classical amyloid fibrils. Despite its high propensity to aggregate, Con-Alz does not show any significant ability to permeabilize vesicles, which for fibrillating proteins is taken to be a key factor in aggregate cytotoxicity and is attributed to oligomers formed at an early stage in the fibrillation process. Physically linking multiple copies of the Aß-peptide may thus sterically restrict Con-Alz against forming cytotoxic oligomers, forcing it instead to adopt a less well-organized assembly of intermeshed polypeptide chains. Copyright © 2010 Elsevier B.V. All rights reserved.

Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

PubMed Central

Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

2003-01-01

We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic. PMID:12614195

How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

PubMed Central

Hytönen, Vesa P; Vogel, Viola

2008-01-01

Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

Embedding the Ni-SOD mimetic Ni-NCC within a polypeptide sequence alters the specificity of the reaction pathway.

PubMed

Krause, Mary E; Glass, Amanda M; Jackson, Timothy A; Laurence, Jennifer S

2013-01-07

The unique metal abstracting peptide asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to dld-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion. Characterization of the NCC sequence within longer peptides with absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies and mass spectrometry (MS) shows that the geometry of metal coordination is maintained, though the electronic properties of the complex are varied to a small extent because of bis-amide, rather than amine/amide, coordination. In addition, both Ni-tripeptide and Ni-pentapeptide complexes have charges of -2. This study demonstrates that the chiral inversion chemistry does not occur when NCC is embedded in a longer polypeptide sequence. Nonetheless, the superoxide scavenging reactivity of the embedded Ni-NCC module is similar to that of the chirally inverted tripeptide complex, which is consistent with a minor change in the reduction potential for the Ni-pentapeptide complex. Together, this suggests that the charge of the complex could affect the SOD activity as much as a change in the primary coordination sphere. In Ni-NCC and other Ni-SOD mimics, changes in chirality, superoxide scavenging activity, and oxidation of the peptide itself all depend on the presence of dioxygen or its reduced derivatives (e.g., superoxide), and the extent to which each of these distinct reactions occurs is ruled by electronic and steric effects that emenate from the organization of ligands around the metal center.

Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

PubMed Central

Mena, Ignacio; Jambrina, Enrique; Albo, Carmen; Perales, Beatriz; Ortín, Juan; Arrese, Marta; Vallejo, Dolores; Portela, Agustín

1999-01-01

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis. PMID:9882320

Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

PubMed

Mercurio, Flavia A; Costantini, Susan; Di Natale, Concetta; Pirone, Luciano; Guariniello, Stefano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Leone, Marilisa

2017-09-01

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains. Copyright © 2017 Elsevier B.V. All rights reserved.

Function of the family-9 and family-22 carbohydrate-binding modules in a modular beta-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B.

PubMed

Zhao, Guangshan; Ali, Ehsan; Araki, Rie; Sakka, Makiko; Kimura, Tetsuya; Sakka, Kazuo

2005-08-01

Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and beta-1,3-1,4-glucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and beta-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially beta-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 degrees C to 75 degrees C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.

Group additivity calculations of the thermodynamic properties of unfolded proteins in aqueous solution: a critical comparison of peptide-based and HKF models.

PubMed

Hakin, A W; Hedwig, G R

2001-02-15

A recent paper in this journal [Amend and Helgeson, Biophys. Chem. 84 (2000) 105] presented a new group additivity model to calculate various thermodynamic properties of unfolded proteins in aqueous solution. The parameters given for the revised Helgeson-Kirkham-Flowers (HKF) equations of state for all the constituent groups of unfolded proteins can be used, in principle, to calculate the partial molar heat capacity, C(o)p.2, and volume, V2(0), at infinite dilution of any polypeptide. Calculations of the values of C(o)p.2 and V2(0) for several polypeptides have been carried out to test the predictive utility of the HKF group additivity model. The results obtained are in very poor agreement with experimental data, and also with results calculated using a peptide-based group additivity model. A critical assessment of these two additivity models is presented.

Isolation and properties of viruses from poultry in Hong Kong which represent a new (sixth) distinct group of avian paramyxoviruses.

PubMed

Shortridge, K F; Alexander, D J; Collins, M S

1980-08-01

Eight viruses isolated in Hong Kong were shown to be serologically related. One was obtained from the tracheal swab of a chicken and four were from cloacal swabs of ducks sampled at a poultry dressing plant. Three isolations were made from samples taken at a duck farm: two from pond water and one from faeces. Representatives of these isolates were shown to be paramyxoviruses but were serologically distinct from other avian and mammalian paramyxoviruses by haemagglutination inhibition and neuraminidase inhibition tests. Slight variations were seen in the properties of three isolates examined in detail. All three were apathogenic for chickens. The structural polypeptides of one isolate, PMV-6/duck/Hong Kong/199/77, were examined by SDS-polyacrylamide gel electrophoresis. Seven polypeptides were detected, with mol. wt. 180000, 76000, 60000, 55000, 51000, 48000 and 40000. The isolates represent a sixth serologically distinct avian paramyxovirus group.

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

PubMed Central

Dever, Thomas E.; Kinzy, Terri Goss; Pavitt, Graham D.

2016-01-01

In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

PubMed Central

Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

2015-01-01

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

Nanocrystalline diamond sensor targeted for selective CRP detection: an ATR-FTIR spectroscopy study.

PubMed

Andersson, Per Ola; Viberg, Pernilla; Forsberg, Pontus; Nikolajeff, Fredrik; Österlund, Lars; Karlsson, Mikael

2016-05-01

Protein immobilization on functionalized fluorine-terminated nanocrystalline (NCD) films was studied by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy using an immobilization protocol developed to specifically bind C-reactive protein (CRP). Using an ATR-FTIR spectroscopy method employing a force-controlled anvil-type configuration, three critical steps of the ex situ CRP immobilization were analyzed. First, the NCD surface was passivated by deposition of a copolymer layer consisting of polyethylene oxide and polypropylene oxide. Second, a synthetic modified polypeptide binder with high affinity to CRP was covalently attached to the polymeric film. Third, CRP dissolved in aqueous buffer in concentrations of 10-20 μg/mL was added on the functionalized NCD surface. Both the amide I and II bands, due to the polypeptide binder and CRP, were clearly observed in ATR-FTIR spectra. CRP amide I bands were extracted from difference spectra and yielded bands that agreed well with the reported amide I band of free (non-bonded) CRP in solution. Thus, our results show that CRP retains its secondary structure when it is attached to the polypeptide binders. Compared to previous IR studies of CRP in solution, about 200 times lower concentration was applied in the present study. Graphical Abstract Direct non-destructive ATR-FTIR analysis of C-reactive protein (CRP) selectively bound to functionalized nanocrystalline diamond (NCD) sensor surface.

Multilayer biomimetics: reversible covalent stabilization of a nanostructured biofilm.

PubMed

Li, Bingyun; Haynie, Donald T

2004-01-01

Designed polypeptides and electrostatic layer-by-layer self-assembly form the basis of promising research in bionanotechnology and medicine on development of polyelectrolyte multilayer films (PEMs). We show that PEMs can be formed from oppositely charged 32mers containing several cysteine residues. The polypeptides in PEMs become cross-linked under mild oxidizing conditions. This mimicking of disulfide (S-S) bond stabilization of folded protein structure confers on the PEMs a marked increase in resistance to film disassembly at acidic pH. The reversibility of S-S bond stabilization of PEMs presents further advantages for controlling physical properties of films, coatings, and other applications involving PEMs.

Residue length and solvation model dependency of elastinlike polypeptides

NASA Astrophysics Data System (ADS)

Bilsel, Mustafa; Arkin, Handan

2010-05-01

We have performed exhaustive multicanonical Monte Carlo simulations of elastinlike polypeptides with a chain including amino acids (valine-proline-glycine-valine-glycine)n or in short (VPGVG)n , where n changes from 1 to 4, in order to investigate the thermodynamic and structural properties. To predict the characteristic secondary structure motifs of the molecules, Ramachandran plots were prepared and analyzed as well. In these studies, we utilized a realistic model where the interactions between all types of atoms were taken into account. Effects of solvation were also simulated by using an implicit-solvent model with two commonly used solvation parameter sets and compared with the vacuum case.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Koushik; Balog, Eva Rose M.; Sista, Prakash

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 themore » 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.« less

Characterization of Ether-à-go-go Channels Present in Photoreceptors Reveals Similarity to IKx, a K+ Current in Rod Inner Segments

PubMed Central

Frings, Stephan; Brüll, Nicole; Dzeja, Claudia; Angele, Albert; Hagen, Volker; Kaupp, U. Benjamin; Baumann, Arnd

1998-01-01

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels. PMID:9524140

Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

NASA Astrophysics Data System (ADS)

Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

1987-10-01

The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

Structure of a Type-1 Secretion System ABC Transporter.

PubMed

Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

2017-03-07

Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

PubMed Central

Oblong, J E; Lamppa, G K

1992-01-01

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet. Images PMID:1385116

Cellobiohydrolase I enzymes

DOEpatents

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

A Nontoxic Polypeptide Oligomer with a Fungicide Potency under Agricultural Conditions Which Is Equal or Greater than That of Their Chemical Counterparts

PubMed Central

Monteiro, Sara; Carreira, Alexandra; Freitas, Regina; Pinheiro, Ana Margarida; Ferreira, Ricardo Boavida

2015-01-01

There are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural conditions. The reasons behind may be multiple where the sensitivity to the Sun UV radiation can come in first place. Here we describe a multifunctional glyco-oligomer with 210 kDa which is mainly composed by a 20 kDa polypeptide termed Blad that has been previously shown to be a stable intermediary product of β-conglutin catabolism. This oligomer accumulates exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Blad-oligomer reveals a plethora of biochemical properties, like lectin and catalytic activities, which are not unusual per si, but are remarkable when found to coexist in the same protein molecule. With this vast range of chemical characteristics, antifungal activity arises almost as a natural consequence. The biological significance and potential technological applications of Blad-oligomer as a plant fungicide to agriculture, its uniqueness stems from being of polypeptidic in nature, and with efficacies which are either equal or greater than the top fungicides currently in the market are addressed. PMID:25849076

Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins

PubMed Central

Gibert, Benjamin; Eckel, Bénédicte; Fasquelle, Lydie; Moulin, Maryline; Bouhallier, Frantz; Gonin, Vincent; Mellier, Gregory; Simon, Stéphanie; Kretz-Remy, Carole; Arrigo, André-Patrick; Diaz-Latoud, Chantal

2012-01-01

Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions. PMID:22238643

Methods for transforming and expression screening of filamentous fungal cells with a DNA library

DOEpatents

Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

2015-06-02

The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

Characterization of the Polypeptides in Varicella Zoster Virus - Infected Cells

DTIC Science & Technology

1984-03-16

DNA binding proteins.. 127 38. Autoradiogram of guanidine hydrochloride wash of DNA cellulose columns 129 Figure Page 32 39. Autoradiogram of P...of purification was seventy-fold 35 1^ with respect to host proteins and the S-methionine or G- glucosamine labeled virions were subjected to SDS... hydrochloride [pH7.5]. 20 mM EDTA, (2 x STE buffer), was used. For electron microscopy pellets were resuspended in 10 mM Tris- hydrochloride [pH 7.5]. 1 inM

Manipulating the membrane penetration mechanism of helical polypeptides via aromatic modification for efficient gene delivery.

PubMed

Zheng, Nan; Song, Ziyuan; Yang, Jiandong; Liu, Yang; Li, Fangfang; Cheng, Jianjun; Yin, Lichen

2017-08-01

The delivery performance of non-viral gene vectors is greatly related to their intracellular kinetics. Cationic helical polypeptides with potent membrane penetration properties and gene transfection efficiencies have been recently developed by us. However, they suffer from severe drawbacks in terms of their membrane penetration mechanisms that mainly include endocytosis and pore formation. The endocytosis mechanism leads to endosomal entrapment of gene cargos, while the charge- and helicity-induced pore formation causes appreciable cytotoxicity at high concentrations. With the attempt to overcome such critical challenges, we incorporated aromatic motifs into the design of helical polypeptides to enhance their membrane activities and more importantly, to manipulate their membrane penetration mechanisms. The aromatically modified polypeptides exhibited higher cellular internalization level than the unmodified analogue by up to 2.5 folds. Such improvement is possibly because aromatic domains promoted the polypeptides to penetrate cell membranes via direct transduction, a non-endocytosis and non-pore formation mechanism. As such, gene cargos were more efficiently delivered into cells by bypassing endocytosis and subsequently avoiding endosomal entrapment, and the material toxicity associated with excessive pore formation was also reduced. The top-performing aromatic polypeptide containing naphthyl side chains at the incorporated content of 20mol% revealed notably higher transfection efficiencies than commercial reagents in melanoma cells in vitro (by 11.7 folds) and in vivo (by 9.1 folds), and thus found potential utilities toward topical gene delivery for cancer therapy. Cationic helical polypeptides, as efficient gene delivery materials, suffer from severe drawbacks in terms of their membrane penetration mechanisms. The main cell penetration mechanisms involved are endocytosis and pore formation. However, the endocytosis mechanism has the limitation of endosomal entrapment of gene cargos, while the charge- and helicity-induced pore formation causes cytotoxicity at high concentrations. To address such critical issues toward the maximization of gene delivery efficiency, we incorporated aromatic domains into helical polypeptides to promote the cell membrane penetrations via direct transduction, which is a non-endocytosis and non-pore formation mechanism. The manipulation of their membrane penetration mechanisms allows gene cargos to be more efficiently delivered by bypassing endocytosis and subsequently avoiding endosomal entrapment. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

PubMed Central

Gopal-Srivastava, R; Mallonee, D H; White, W B; Hylemon, P B

1990-01-01

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium. Images PMID:2376563

Tailored design of multifunctional and programmable pH-responsive self-assembling polypeptides as drug delivery nanocarrier for cancer therapy.

PubMed

Wang, Tzu-Wei; Yeh, Chia-Wei; Kuan, Chen-Hsiang; Wang, Li-Wen; Chen, Liang-Hsin; Wu, Hsi-Chin; Sun, Jui-Shen

2017-08-01

Breast cancer has become the second leading cause of cancer-related mortality in female wherein more than 90% of breast cancer-related death results from cancer metastasis to distant organs at advanced stage. The purpose of this study is to develop biodegradable nanoparticles composed of natural polypeptides and calcium phosphate (CaP) with sequential pH-responsivity to tumor microenvironments for active targeted drug delivery. Two different amphiphilic copolymers, poly(ethylene glycol) 3400 -aconityl linkage-poly(l-glutamic acid) 15 -poly(l-histidine) 10 -poly(l-leucine) 10 and LyP1-poly(ethylene glycol) 1100 -poly(l-glutamic acid) 15 -poly(l-histidine) 10 -poly(l-leucine) 10 , were exploited to self-assemble into micelles in aqueous phase. The bio-stable nanoparticles provide three distinct functional domains: the anionic PGlu shell for CaP mineralization, the protonation of PHis segment for facilitating anticancer drug release at target site, and the hydrophobic core of PLeu for encapsulation of anticancer drugs. Furthermore, the hydrated PEG outer corona is used for prolonging circulation time, while the active targeting ligand, LyP-1, is served to bind to breast cancer cells and lymphatic endothelial cells in tumor for inhibiting metastasis. Mineralized DOX-loaded nanoparticles (M-DOX NPs) efficiently prevent the drug leakage at physiological pH value and facilitate the encapsulated drug release at acidic condition when compared to DOX-loaded nanoparticles (DOX NPs). M-DOX NPs with LyP-1 targeting ligand effectively accumulated in MDA-MB-231 breast cancer cells. The inhibition effect on cell proliferation also enhances with time, illustrating the prominent anti-tumor efficacy. Moreover, the in vitro metastatic inhibition model shows the profound inhibition effect of inhibitory nanoparticles. In brief, this self-assembling peptide-based drug delivery nanocarrier with multifunctionality and programmable pH-sensitivity is of great promise and potential for anti-cancer therapy. This tailored-design polypeptide-based nanoparticles with self-assembling and programmable stimulus-responsive properties enable to 1) have stable pH in physiological value with a low level of drug loss and effectively release the encapsulated drug with pH variations according to the tumor microenvironment, 2) enhance targeting ability to hard-to-treat breast cancer cells and activate endothelial cells (tumor region), 3) significantly inhibit the growth and prevent from malignant metastasis of cancer cells in consonance with promising anti-tumor efficacy, and 4) make tumors stick to localized position so that these confined solid tumors can be more accessible by different treatment modalities. This work contributes to designing a programmable pH-responsive drug delivery system based on the tailor-designed polypeptides. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Receptors for VIP and PACAP in guinea pig cerebral cortex: effects on cyclic AMP synthesis and characterization by 125I-VIP binding.

PubMed

Zawilska, Jolanta B; Dejda, Agnieszka; Niewiadomski, Pawel; Gozes, Illana; Nowak, Jerzy Z

2005-01-01

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of 125I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of 125I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a B(max) of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of 125I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) > or = PACAP38 approximately PACAP27 approximately guinea pig VIP (gpVIP) > or = mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of 125I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific 125I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [3H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 microM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 microM, produced net increases in cAMP production in the range of 8-9% conversion, whereas 5 microM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E*

PubMed Central

Siddiqui, Shoib; Schwarz, Flavio; Springer, Stevan; Khedri, Zahra; Yu, Hai; Deng, Lingquan; Verhagen, Andrea; Naito-Matsui, Yuko; Jiang, Weiping; Kim, Daniel; Zhou, Jie; Ding, Beibei; Chen, Xi; Varki, Nissi; Varki, Ajit

2017-01-01

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer. PMID:27920204

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.

PubMed

Athwal, G S; Lombardo, C R; Huber, J L; Masters, S C; Fu, H; Huber, S C

2000-04-01

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

The gamma irradiation effects on structural and optical properties of silk fibroin/HPMC blend films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shetty, G. Rajesha; Rao, B. Lakshmeesha; Gowda, Mahadeva

In this paper the structural, chemical and optical properties of gamma irradiated silk fibroin/Hydroxypropyl methyl cellulose (SF-HPMC) blend films were studied using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and UV-visible spectroscopy. The results indicate that the gamma radiation did not affect significantly the primary structure of polypeptide arrangement in the blend films. But the optical properties of the blends changed with gamma irradiation dosage.

Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal.

PubMed

Jain, Rinku; Hao, Bing; Liu, Ren-Peng; Chan, Michael K

2005-04-06

E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

PubMed

Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

2014-03-01

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

Crystal structure of a chimaeric bacterial glutamate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.

2016-05-23

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P) +as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD +versusNADP +, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies,more » as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP +cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.« less

Characterisation of interaction between food colourant allura red AC and human serum albumin: multispectroscopic analyses and docking simulations.

PubMed

Wu, Di; Yan, Jin; Wang, Jing; Wang, Qing; Li, Hui

2015-03-01

Binding interaction of human serum albumin (HSA) with allura red AC, a food colourant, was investigated at the molecular level through fluorescence, ultraviolet-visible, circular dichroism (CD) and Raman spectroscopies, as well as protein-ligand docking studies to better understand the chemical absorption, distribution and transportation of colourants. Results show that allura red AC has the ability to quench the intrinsic fluorescence of HSA through static quenching. The negative values of the thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bond and van der Waals forces are dominant in the binding between the food colourant and HSA. The CD and Raman spectra showed that the binding of allura red AC to HSA induces the rearrangement of the carbonyl hydrogen-bonding network of polypeptides, which changes the HSA secondary structure. This colourant is bound to HSA in site I, and the binding mode was further analysed with the use of the CDOCKER algorithm in Discovery Studio. Copyright © 2014 Elsevier Ltd. All rights reserved.

The interaction of Clostridium perfringens enterotoxin with receptor claudins

PubMed Central

Shrestha, Archana; Uzal, Francisco A.; McClane, Bruce A.

2016-01-01

Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery. PMID:27090847

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

PubMed

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

PubMed

Means, A L; Slansky, J E; McMahon, S L; Knuth, M W; Farnham, P J

1992-03-01

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary.

The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

PubMed Central

Means, A L; Slansky, J E; McMahon, S L; Knuth, M W; Farnham, P J

1992-01-01

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary. Images PMID:1545788

Polypeptide Synthesis in Simian Virus 5-Infected Cells

PubMed Central

Peluso, Richard W.; Lamb, Robert A.; Choppin, Purnell W.

1977-01-01

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F0) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F1 and F2. No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed. Images PMID:196101

Method for creating polynucleotide and polypeptide sequences

NASA Technical Reports Server (NTRS)

Arnold, Frances (Inventor); Volkov, Alexander (Inventor); Shao, Zhixin (Inventor)

2003-01-01

The invention provides methods for evolving a polynucleotide toward acquisition of a desired property. Such methods entail incubating a population of parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes. The heteroduplexes are then exposed to a cellular DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants. The resulting polynucleotides are then screened or selected for the desired property.

Interactions of urea with native and unfolded proteins: a volumetric study.

PubMed

Son, Ikbae; Shek, Yuen Lai; Tikhomirova, Anna; Baltasar, Eduardo Hidalgo; Chalikian, Tigran V

2014-11-26

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

PubMed Central

Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

2018-01-01

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch

The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-IImore » binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.« less

A Promising Therapeutic Target for Metabolic Diseases: Neuropeptide Y Receptors in Humans.

PubMed

Yi, Min; Li, Hekai; Wu, Zhiye; Yan, Jianyun; Liu, Qicai; Ou, Caiwen; Chen, Minsheng

2018-01-01

Human neuropeptide Y (hNPY) is one of the most widely expressed neurotransmitters in the human central and peripheral nervous systems. It consists of 36 highly conserved amino acid residues, and was first isolated from the porcine hypothalamus in 1982. While it is the most recently discovered member of the pancreatic polypeptide family (which includes neuropeptide Y, gut-derived hormone peptide YY, and pancreatic polypeptide), NPY is the most abundant peptide found in the mammalian brain. In order to exert particular functions, NPY needs to bind to the NPY receptor to activate specific signaling pathways. NPY receptors belong to the class A or rhodopsin-like G-protein coupled receptor (GPCR) family and signal via cell-surface receptors. By binding to GPCRs, NPY plays a crucial role in various biological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. Abnormal regulation of NPY is involved in the development of a wide range of diseases, including obesity, hypertension, atherosclerosis, epilepsy, metabolic disorders, and many cancers. Thus far, five receptors have been cloned from mammals (Y1, Y2, Y4, Y5, and y6), but only four of these (hY1, hY2, hY4, and hY5) are functional in humans. In this review, we summarize the structural characteristics of human NPY receptors and their role in metabolic diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Two novel Mesocestoides vogae fatty acid binding proteins--functional and evolutionary implications.

PubMed

Alvite, Gabriela; Canclini, Lucía; Corvo, Ileana; Esteves, Adriana

2008-01-01

This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corresponding polypeptide chains share 62% identical residues and overall 90% similarity according to CLUSTALX default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3/FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon-intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified.

Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eid, Rawan; Department of Biology, Queen's University, Kingston, Ontario; Boucher, Eric

Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomycesmore » cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. - Highlights: • Human ferritin, heavy polypeptide 1 (FTH1) was identified as a suppressor of the pro-apoptotic Bax in yeast. • Based on its similarity to ferritin we examined Rgi1p/YER067W for potential ferritin like functions. • Like human H-ferritin, RGI1 confers increased resistance to apoptotic inducing stresses in yeast. • Rgi1p is a pro-survival protein that is not a ferritin but that may play a role in iron metabolism of yeast.« less

Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains.

PubMed

Petitdemange, Rosine; Garanger, Elisabeth; Bataille, Laure; Bathany, Katell; Garbay, Bertrand; Deming, Timothy J; Lecommandoux, Sébastien

2017-05-17

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.

Macromolecules Vis-a-Vis the Traditions of Chemistry

ERIC Educational Resources Information Center

Flory, Paul J.

1973-01-01

Summarizes the history of concepts concerning the molecular nature of polymers, involving the carbon chain theory, graphic formula, polycondensation, colloidal properties, polypeptide hypothesis, secondary aggregation, and Watson-Crick model. Indicates that macromolecular science should be accommodated within the discipline of molecular science…

Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai

2012-02-21

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology ofmore » the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.« less

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins

NASA Astrophysics Data System (ADS)

Reinach, Fernando C.; Nagai, Kiyoshi; Kendrick-Jones, John

1986-07-01

The regulatory light chains, small polypeptides located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation. The demonstration that the regulatory light chains on scallop myosin can be replaced by light chains from other myosins has allowed us to compare the functional capabilities of different light chains1, but has not enabled us to probe the role of features, such as the Ca2+/Mg2+ binding site, that are common to all of them. Here, we describe the use of site-directed mutagenesis to study the function of that site. We synthesized the chicken skeletal myosin light chain in Escherichia coli and constructed mutants with substitutions within the Ca2+/Mg2+ binding site. When the aspartate residues at the first and sixth Ca2+ coordination positions are replaced by uncharged alanines, the light chains have a reduced Ca2+ binding capacity but still bind to scallop myosin with high affinity. Unlike the wild-type skeletal light chain which inhibits myosin interaction with actin, the mutants activate it. Thus, an intact Ca2+/Mg2+ binding site in the N-terminal region of the light chain is essential for regulating the interaction of myosin with actin.

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A.

PubMed Central

Cole, C N; Tornow, J; Clark, R; Tjian, R

1986-01-01

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains. Images PMID:3003386

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strittmatter, S.M.; Snyder, S.H.

We demonstrate that (3H)captopril selectively labels angiotensin converting enzyme (EC 3.14.15.1) (ACE) and employ this technique to probe enzyme-inhibitor interactions. (3H)Captopril binding sites copurify with ACE activity from rat lung or rat brain. At each stage of the purification the Vmax/Bmax ratio, or kcat is 17,000 min-1 with hippuryl-L-histidyl-L-leucine as substrate. The specificity of (3H)captopril binding is apparent in the similar pharmacologic profile of inhibition in crude and pure enzyme preparations. Furthermore, binding sites and enzyme activity comigrate in gel filtration and sucrose gradient sedimentation experiments. Equilibrium analysis of (3H)captopril binding to purified ACE reveals a Bmax of 6 nmol/mgmore » of protein (KD = 2 nM), demonstrating the presence of one inhibitor binding site per polypeptide chain. The kinetics of (3H)captopril binding are characterized by monophasic association and dissociation rate constants of 0.026 nM-1 min-1 and 0.034 min-1, respectively. The affinity of ACE for both (3H) captopril and enalaprilat is greater at 37 degrees than at 0 degree, demonstrating that these interactions are entropically driven, perhaps by an isomerization of the enzyme molecule. The ionic requirements for (3H)captopril binding and substrate catalysis differ. Chloride and bromide ion, but not fluoride, are about 100-fold more potent stimulators of binding than catalysis. When the active site Zn2+ ion is replaced by Co2+, catalysis was stimulated 2-fold, whereas binding activity was decreased by 70%.« less

Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode

PubMed Central

Yang, Hyun-Jong; Shin, Joo-Ho; Diaz-Camacho, Sylvia Paz; Nawa, Yukifumi; Kang, Insug; Kong, Yoon

2012-01-01

Background Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. Methodology/Principal Findings We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol. Conclusions/Significance The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design. PMID:23150743

Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides.

PubMed

Li, Nan K; Roberts, Stefan; Quiroz, Felipe Garcia; Chilkoti, Ashutosh; Yingling, Yaroslava G

2018-04-30

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature ( T t ). However, poly(VPGVG) resolubilizes upon cooling below its T t , whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the T t . The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above T t , where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).

Interactions between Kar2p and Its Nucleotide Exchange Factors Sil1p and Lhs1p Are Mechanistically Distinct*

PubMed Central

Hale, Sarah J.; Lovell, Simon C.; de Keyzer, Jeanine; Stirling, Colin J.

2010-01-01

Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p. In contrast, the IIB domain of Kar2p is sufficient for binding of Sil1p, and point mutations within IIB specifically blocked Sil1p-dependent activation while remaining competent for activation by Lhs1p. Taken together, these results demonstrate that the interactions between Kar2p and its two nucleotide exchange factors can be functionally resolved and are thus mechanistically distinct. PMID:20430899

Potential New Ligand Systems for Binding Uranyl Ions in Seawater Environments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arnold, John

2014-12-13

Work began this quarter on a new project involving a combined computational and biosynthetic approach to selective recognition of uranyl ion in aqueous solution. This project exploits the results of computational studies to discover new ligand classes. Synthetic studies will follow to generate target systems for uranyl binding and determination of binding constants. The process will be iterative, with results from computation informing synthesis, and vice versa. The theme of the ligand classes to be examined initially will be biologically based. New phosphonate-containing α-amino acid N-carboxyanhydride (NCA) monomers were used recently to prepare well-defined phosphonate-containing poly-peptides and block copolypeptides. Ourmore » first approach is to utilize these phosphate- and phosphonate-containing NCAs for the coordination of uranyl. The work includes the laboratory-scale preparation of a series of NCAs and the full thermodynamic and spectroscopic characterization of the resulting uranyl complexes. We are also evaluating the sequestering activity in different physiological and environmental conditions of these copolymers as well as their biodegradability.« less

Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain).

PubMed

Zeng, Xianglan; Ye, Haihui; Yang, Ya'nan; Wang, Guizhong; Huang, Huiyang

2013-03-01

Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab.

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lira, C.B.B.; Instituto de Biologia, UNICAMP, Campinas, SP; Siqueira Neto, J.L.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA andmore » to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.« less

Shape-Persistent, Thermoresponsive Polypeptide Brushes Prepared by Vapor Deposition Surface-Initiated Ring-Opening Polymerization of α-Amino Acid N -Carboxyanhydrides

DOE PAGES

Shen, Yong; Desseaux, Solenne; Aden, Bethany; ...

2015-04-20

We report that surface-grafting thermoresponsive polymers allows the preparation of thin polymer brush coatings with surface properties that can be manipulated by variation of temperature. In most instances, thermoresponsive polymer brushes are produced using polymers that dehydrate and collapse above a certain temperature. This report presents the preparation and properties of polymer brushes that show thermoresponsive surface properties, yet are shape-persistent in that they do not undergo main chain collapse. The polymer brushes presented here are obtained via vapor deposition surface-initiated ring-opening polymerization (SI-ROP) of γ-di- or tri(ethylene glycol)-modified glutamic acid N-carboxyanhydrides. Vapor deposition SI-ROP of γ-di- or tri(ethylene glycol)-modifiedmore » L- or D-glutamic acid N-carboxyanhydrides affords helical surface-tethered polymer chains that do not show any changes in secondary structure between 10 and 70 °C. QCM-D experiments, however, revealed significant dehydration of poly(γ-(2-(2-methoxyethoxy)ethyl)-l-glutamate) (poly(L-EG 2-Glu)) brushes upon heating from 10 to 40 °C. At the same time, AFM and ellipsometry studies did not reveal significant variations in film thickness over this temperature range, which is consistent with the shape-persistent nature of these polypeptide brushes and indicates that the thermoresponsiveness of the films is primarily due to hydration and dehydration of the oligo(ethylene glycol) side chains. The results we present here illustrate the potential of surface-initiated NCA ring-opening polymerization to generate densely grafted assemblies of polymer chains that possess well-defined secondary structures and tunable surface properties. These polypeptide brushes complement their conformationally unordered counterparts that can be generated via surface-initiated polymerization of vinyl-type monomers and represent another step forward to biomimetic surfaces and interfaces.« less

Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

PubMed Central

Wallace, B; Yang, Y J; Hong, J S; Lum, D

1990-01-01

A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system. Images PMID:1971622

The Importance of a Gatekeeper Residue on the Aggregation of Transthyretin

PubMed Central

Sant'Anna, Ricardo; Braga, Carolina; Varejão, Nathalia; Pimenta, Karinne M.; Graña-Montes, Ricardo; Alves, Aline; Cortines, Juliana; Cordeiro, Yraima; Ventura, Salvador; Foguel, Debora

2014-01-01

Protein aggregation into β-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26–57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26–57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility. PMID:25086037

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

PubMed

Doern, Adam; Cao, Xianjun; Sereno, Arlene; Reyes, Christopher L; Altshuler, Angelina; Huang, Flora; Hession, Cathy; Flavier, Albert; Favis, Michael; Tran, Hon; Ailor, Eric; Levesque, Melissa; Murphy, Tracey; Berquist, Lisa; Tamraz, Susan; Snipas, Tracey; Garber, Ellen; Shestowsky, William S; Rennard, Rachel; Graff, Christilyn P; Wu, Xiufeng; Snyder, William; Cole, Lindsay; Gregson, David; Shields, Michael; Ho, Steffan N; Reff, Mitchell E; Glaser, Scott M; Dong, Jianying; Demarest, Stephen J; Hariharan, Kandasamy

2009-04-10

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Luminescent macrocyclic lanthanide complexes

DOEpatents

Raymond, Kenneth N; Corneillie, Todd M; Xu, Jide

2014-05-20

The present invention provides a novel class of macrocyclic compounds as well as complexes formed between a metal (e.g., lanthanide) ion and the compounds of the invention. Preferred complexes exhibit high stability as well as high quantum yields of lanthanide ion luminescence in aqueous media without the need for secondary activating agents. Preferred compounds incorporate hydroxy-isophthalamide moieties within their macrocyclic structure and are characterized by surprisingly low, non-specific binding to a variety of polypeptides such as antibodies and proteins as well as high kinetic stability. These characteristics distinguish them from known, open-structured ligands.

Structure of rat acidic fibroblast growth factor at 1.4 Å resolution

PubMed Central

Kulahin, Nikolaj; Kiselyov, Vladislav; Kochoyan, Arthur; Kristensen, Ole; Kastrup, Jette Sandholm; Berezin, Vladimir; Bock, Elisabeth; Gajhede, Michael

2007-01-01

Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 Å resolution X-ray structure of rat FGF1 is presented. Two molecules are present in the asymmetric unit of the crystal and they coordinate a total of five sulfate ions. The structures of human, bovine and newt FGF1 have been published previously. Human and rat FGF1 are found to have very similar structures. PMID:17277441

Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis.

PubMed

Boccafoschi, Francesca; Ramella, Martina; Sibillano, Teresa; De Caro, Liberato; Giannini, Cinzia; Comparelli, Roberto; Bandiera, Antonella; Cannas, Mario

2015-03-01

The replacement of diseased tissues with biological substitutes with suitable biomechanical properties is one of the most important goal in tissue engineering. Collagen represents a satisfactory choice for scaffolds. Unfortunately, the lack of elasticity represents a restriction to a wide use of collagen for several applications. In this work, we studied the effect of human elastin-like polypeptide (HELP) as hybrid collagen-elastin matrices. In particular, we studied the biomechanical properties of collagen/HELP scaffolds considering several components involved in ECM remodeling (elastin, collagen, fibrillin, lectin-like receptor, metalloproteinases) and cell phenotype (myogenin, myosin heavy chain) with particular awareness for vascular tissue engineering applications. Elastin and collagen content resulted upregulated in collagen-HELP matrices, even showing an improved structural remodeling through the involvement of proteins to a ECM remodeling activity. Moreover, the hybrid matrices enhanced the contractile activity of C2C12 cells concurring to improve the mechanical properties of the scaffold. Finally, small-angle X-ray scattering analyses were performed to enable a very detailed analysis of the matrices at the nanoscale, comparing the scaffolds with native blood vessels. In conclusion, our work shows the use of recombinant HELP, as a very promising complement able to significantly improve the biomechanical properties of three-dimensional collagen matrices in terms of tensile stress and elastic modulus. © 2014 Wiley Periodicals, Inc.

Targeted polypeptide degradation

DOEpatents

Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

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.

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site.

PubMed

Ogawa, Haruo; Qiu, Yue; Philo, John S; Arakawa, Tsutomu; Ogata, Craig M; Misono, Kunio S

2010-03-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

Reversibly Bound Chloride in the Atrial Natriuretic Peptide Receptor Hormone Binding Domain: Possible Allosteric Regulation and a Conserved Structural Motif for the Chloride-binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogawa, H.; Qiu, Y; Philo, J

2010-01-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. Amore » new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.« less

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: Possible allosteric regulation and a conserved structural motif for the chloride-binding site

PubMed Central

Ogawa, Haruo; Qiu, Yue; Philo, John S; Arakawa, Tsutomu; Ogata, Craig M; Misono, Kunio S

2010-01-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(−)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(−) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(−) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis. PMID:20066666

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

PubMed

Gustafsson, Mattias C U; Lannergård, Jonas; Nilsson, O Rickard; Kristensen, Bodil M; Olsen, John E; Harris, Claire L; Ufret-Vincenty, Rafael L; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

PubMed Central

Kristensen, Bodil M.; Olsen, John E.; Harris, Claire L.; Ufret-Vincenty, Rafael L.; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems. PMID:23637608

Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions.

PubMed

Read, G Sullivan; Patterson, Mary

2007-02-01

The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

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

PubMed

Ahmed, Marya

2017-10-24

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

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.

Doxycycline exerted neuroprotective activity by enhancing the activation of neuropeptide GPCR PAC1.

PubMed

Yu, Rongjie; Zheng, Lijun; Cui, Yue; Zhang, Huahua; Ye, Heng

2016-04-01

Doxycycline has significant neuroprotective effect with anti-inflammatory and anti-apoptotic activity. We found for the first time that doxycycline specially promoted the proliferation of Chinese hamster ovary (CHO) cells with high expression of neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) preferring G protein-coupled receptor (GPCR), PACAP receptor 1(PAC1) and induced the internalization of PAC1 tagged with yellow fluorescent protein (YFP) indicating doxycycline interacted with PAC1. The homology modeling of PAC1 and molecular docking of doxycycline with PAC1 showed the theoretical binding of doxycycline to PAC1 at the site where PACAP(30-37) recognized. The competition binding assay and PAC1 site-specific mutation of Asp116, which formed two hydrogen bonds with Dox, confirmed the binding of doxycycline to PAC1 imitating PACAP(30-37). Doxycycline (100 ng/mL) significantly promoted the proliferative activities of vasoactive intestinal polypeptide (VIP) and oligopeptide HSDGIF responsible for the activation of PAC1 in PAC1-CHO cells, indicating that doxycycline facilitated the binding and the activation of PAC1 imitating PACAP(28-38). In Neuro2a cells with endogenous expression of PAC1 and its ligands, doxycycline not only promoted the proliferation of Neuro2a cells but also protected the cells from scopolamine induced apoptosis, which was inhibited by cAMP-PKA signal pathway inhibitor H-89, PAC1 shRNA or PACAP antagonist PACAP(6-38). The in vivo study showed long-term treatment with doxycycline (100ug/kg) had significant effect against scopolamine induced amnesia, and the synergetic anti-apoptotic, anti-oxidative and neuroprotective effect of doxycycline with VIP was more efficient than doxycycline alone or VIP alone, indicating doxycycline enhanced the activation of PAC1 in vivo effectively. Furthermore, doxycycline analogue minocycline also had similar theoretically binding site on PAC1 to doxycycline and displayed corresponding similar activity on PAC1 to doxycycline. All these results confirmed for the first time that doxycycline specially targeted PAC1 imitating PACAP(30-37) and acted as an enhancer by facilitating the subsequent ligand binding and the activation of PAC1. The confirmation of PAC1 as a novel molecular target of doxycycline and the novel mechanism by which doxycycline enhances the activity of PAC1 will help further clinical development of doxycycline as novel therapy for nervous system diseases such as neurodegenerative diseases targeting PAC1. Copyright © 2015 Elsevier Ltd. All rights reserved.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

NASA Technical Reports Server (NTRS)

Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

2000-01-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

PubMed

Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

2000-10-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Identification of globular mechanochemical heads of kinesin.

PubMed

Scholey, J M; Heuser, J; Yang, J T; Goldstein, L S

1989-03-23

Kinesin is a mechanoenzyme which uses energy liberated from ATP hydrolysis to transport particles towards the 'plus ends' of microtubules. The enzyme consists of two polypeptide heavy chains of relative molecular mass (Mr) approximately 110,000-140,000 (110K-140K) plus copurifying light chains; these polypeptides are arranged in a structure consisting of two globular heads attached to a fibrous stalk which terminates in a 'feathered' tail. Here we report that a function-disrupting monoclonal antikinesin, which binds to the 45K fragment of the kinesin heavy chain, recognizes an epitope located towards the N-terminal end of the heavy chain, and decorates the two globular heads lying at one end of the intact molecules (one antibody per head). The results show that the two heavy chains of native kinesin are arranged in parallel, and that the 45K fragments, which display nucleotide-sensitive interactions with microtubules, represent mechanochemical 'heads' located at the N-terminal regions of the heavy chains. Thus, it is likely that the kinesin heads are analogous to the subfragment-1 domains of myosin.

Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes

PubMed Central

Pfister, Klaus; Steinback, Katherine E.; Gardner, Gary; Arntzen, Charles J.

1981-01-01

2-Azido-4-ethylamino-6-isopropylamino-s-triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido[14C]atrazine resulted in the covalent attachment of radioactive inhibitor to thylakoid membranes isolated from pea seedlings and from a triazine-susceptible biotype of the weed Amaranthus hybridus. No covalent binding of azido-atrazine was observed for thylakoid membranes isolated from a naturally occurring triazine-resistant biotype of A. hybridus. Analysis of thylakoid polypeptides from both the susceptible and resistant A. hybridus biotypes by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by fluorography to locate 14C label, demonstrated specific association of the azido[14C]atrazine with polypeptides of the 34- to 32-kilodalton size class in susceptible but not in resistant membranes. Images PMID:16592984

The Ribosome-Bound Chaperones RAC and Ssb1/2p Are Required for Accurate Translation in Saccharomyces cerevisiae

PubMed Central

Rakwalska, Magdalena; Rospert, Sabine

2004-01-01

The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides. PMID:15456889

The ribosome-bound chaperones RAC and Ssb1/2p are required for accurate translation in Saccharomyces cerevisiae.

PubMed

Rakwalska, Magdalena; Rospert, Sabine

2004-10-01

The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

PubMed

Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

2016-12-01

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

PubMed Central

Tamura, A; Ohashi, N; Urakami, H; Takahashi, K; Oyanagi, M

1985-01-01

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera. Images PMID:3922893

Appalachian spring: variations on ancient gastro-entero-pancreatic themes in New World mammals.

PubMed

Seino, S; Blackstone, C D; Chan, S J; Whittaker, J; Bell, G I; Steiner, D F

1988-07-01

Studies of guinea pig genomic and/or cDNA clones encoding the gastro-entero-pancreatic (GEP) hormones--insulin, glucagon and pancreatic polypeptide--as well as portions of the insulin receptor, are described. Multiple clustered substitutions (localized rapid mutation acceptance) altering the biological properties of both insulin and glucagon have been revealed, but this does not appear to be the case with either pancreatic polypeptide or those regions of guinea pig insulin receptor cDNAs that have been examined thus far. These findings suggest that novel selective pressures operative in the New World environment, in which these animals evolved in isolation from Old World mammalian species, have led to altered solutions to problems related to the regulation of growth and carbohydrate metabolism.

Surface structural conformations of fibrinogen polypeptides for improved biocompatibility.

PubMed

Yaseen, Mohammed; Zhao, Xiubo; Freund, Amy; Seifalian, Alexander M; Lu, Jian R

2010-05-01

This work reports on how incorporation of silica nanocages into poly(urethane) copolymers (PU) affects conformational orientations of adsorbed fibrinogen and how different surfaces subsequently influenced HeLa cell attachment and proliferation. Incorporation of 2 wt% silica nanocages into poly(urethane) (PU4) substantially altered the surface topography of the films and some 50% of the surface was covered with the nanocages due to their preferential exposure. AFM studies revealed the deposition of a dense protein network on the soft polymeric domains of PU4 and much reduced fibrinogen adsorption on the hard nanocage domains. As on the bare SiO(2) control surface, fibrinogen molecules adsorbed on top of the hard nanocages mainly took the dominant trinodular structures in monomeric and dimeric forms. In addition, net positively charged long alpha chains were prone to being hidden beneath the D domains whilst gamma chains predominantly remained exposed. Dynamic interfacial adsorption as probed by spectroscopic ellipsometry revealed fast changes in interfacial conformation induced by electrostatic interactions between different segments of fibrinogen and the surface, consistent with the AFM imaging. On the PU surfaces without nanocage incorporation (PUA), however, adsorbed fibrinogen molecules formed beads-like chain networks, consistent with the structure featured on the soft PU4 domains, showing very different effects of surface chemical nature. Monoclonal antibodies specific to the alpha and gamma chains showed reduced alpha but increased gamma chain binding at the silicon oxide control and PU4 surfaces, whilst on the PUA, C18 and amine surfaces (organic surface controls) the opposite binding trend was detected with alpha chain binding dominant, showing different fibrinogen conformations. Cell attachment studies revealed differences in cell attachment and proliferation, consistent with the different polypeptide conformations on the two types of surfaces, showing a strong preference to the extent of exposure of gamma chains. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

Cysteine Scanning Mutagenesis of Transmembrane Domain 10 in Organic Anion Transporting Polypeptide 1B1

PubMed Central

2015-01-01

Organic anion transporting polypeptide (OATP) 1B1 is an important drug transporter expressed in human hepatocytes. Previous studies have indicated that transmembrane (TM) domain 2, 6, 8, 9, and in particular 10 might be part of the substrate binding site/translocation pathway. To explore which amino acids in TM10 are important for substrate transport, we mutated 34 amino acids individually to cysteines, expressed them in HEK293 cells, and determined their surface expression. Transport activity of the two model substrates estrone-3-sulfate and estradiol-17β-glucuronide as well as of the drug substrate valsartan for selected mutants was measured. Except for F534C and F537C, all mutants were expressed at the plasma membrane of HEK293 cells. Mutants Q541C and A549C did not transport estradiol-17β-glucuronide and showed negligible estrone-3-sulfate transport. However, A549C showed normal valsartan transport. Pretreatment with the anionic and cell impermeable sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) affected the transport of each substrate differently. Pretreatment of L545C abolished estrone-3-sulfate uptake almost completely, while it stimulated estradiol-17β-glucuronide uptake. Further analyses revealed that mutant L545C in the absence of MTSES showed biphasic kinetics for estrone-3-sulfate that was converted to monophasic kinetics with a decreased apparent affinity, explaining the previously seen inhibition. In contrast, the apparent affinity for estradiol-17β-glucuronide was not changed by MTSES treatment, but the Vmax value was increased about 4-fold, explaining the previously seen stimulation. Maleimide labeling of L545C was affected by preincubation with estrone-3-sulfate but not with estradiol-17β-glucuronide. These results strongly suggest that L545C is part of the estrone-3-sulfate binding site/translocation pathway but is not directly involved in binding/translocation of estradiol-17β-glucuronide. PMID:24673529

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.

Instability of monoclonal myeloma protein may be identified as susceptibility to penetration and binding by newly synthesized Congo red derivatives.

PubMed

Spólnik, Paweł; Konieczny, Leszek; Piekarska, Barbara; Rybarska, Janina; Stopa, Barbara; Zemanek, Grzegorz; Król, Marcin; Roterman, Irena

2004-06-01

Monoclonal myeloma proteins often have an abnormal, unstable structure, and tend to aggregate with fatal clinical consequences. A method for early clinical identification of this aggregation tendency is impatiently awaited. This work proposes the use of supramolecular dyes as specific ligands to reveal protein instability. Disclosure of excessive polypeptide chain flexibility in unstable monoclonal proteins, leading to increased susceptibility to penetration by foreign compounds, appeared possible when new supramolecular Congo red-derived dyes with different protein-binding capabilities were used for complexation. Two basic protein instability levels, local and global, were differentiated by comparing the extent of protein loading with dye and the subsequent electrophoretic migration rate of the complexes. A simple electrophoretic test is proposed for assessment of the instability of monoclonal proteins in clinical conditions.

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

PubMed Central

Cai, M.; Huang, Y.; Caffrey, M.; Zheng, R.; Craigie, R.; Clore, G. M.; Gronenborn, A. M.

1998-01-01

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex. PMID:9865962

DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harford, N.; De Wilde, M.

1987-05-19

A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce anmore » immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.« less

Polypeptides having laccase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides 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.

Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains.

PubMed

Hayes, Michael L; Giang, Karolyn; Berhane, Beniam; Mulligan, R Michael

2013-12-20

Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins are required as RNA binding specificity determinants in the RNA editing mechanism. Bioinformatic analysis has shown that most of the Arabidopsis PPR proteins necessary for RNA editing events include a C-terminal portion that shares structural characteristics with a superfamily of deaminases. The DYW deaminase domain includes a highly conserved zinc binding motif that shares characteristics with cytidine deaminases. The Arabidopsis PPR genes, ELI1 and DOT4, both have DYW deaminase domains and are required for single RNA editing events in chloroplasts. The ELI1 DYW deaminase domain was expressed as a recombinant protein in Escherichia coli and was shown to bind two zinc atoms per polypeptide. Thus, the DYW deaminase domain binds a zinc metal ion, as expected for a cytidine deaminase, and is potentially the catalytic component of an editing complex. Genetic complementation experiments demonstrate that large portions of the DYW deaminase domain of ELI1 may be eliminated, but the truncated genes retain the ability to restore editing site conversion in a mutant plant. These results suggest that the catalytic activity can be supplied in trans by uncharacterized protein(s) of the editosome.

Control of Ion Selectivity in LeuT: Two Na+ Binding Sites with two different mechanisms

PubMed Central

Noskov, Sergei Y.; Roux, Benoît

2016-01-01

The x-ray structure of LeuT, a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporter, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion-binding sites, NA1 and NA2, which are highly selective for Na+. Extensive all-atom free energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na+ over K+ or Li+, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In site NA1, selectivity for Na+ over K+ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In site NA2, which comprises only neutral ligands, selectivity for Na+ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the poly-peptide chain surrounding the ion according to a snug-fit mechanism. PMID:18280500

The Interface between Catalytic and Hemopexin Domains in Matrix Metalloproteinase-1 Conceals a Collagen Binding Exosite*

PubMed Central

Arnold, Laurence H.; Butt, Louise E.; Prior, Stephen H.; Read, Christopher M.; Fields, Gregg B.; Pickford, Andrew R.

2011-01-01

Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe301, Val319, and Asp338 in collagen binding. Intriguingly, Phe301 is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity. PMID:22030392

Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

PubMed

Pessler, F; Pendergrast, P S; Hernandez, N

1997-07-01

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode

PubMed Central

Wagner, Tristan; Ermler, Ulrich; Shima, Seigo

2016-01-01

In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA–H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change. PMID:27324530

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

PubMed

Wieczorek, Anna; McHenry, Charles S

2006-05-05

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Totoritis, Rachel; Duraiswami, Chaya; Taylor, Amy N.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes inmore » time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.« less

Solvent mimicry with methylene carbene to probe protein topography.

PubMed

Gómez, Gabriela Elena; Monti, José Luis E; Mundo, Mariana Rocío; Delfino, José María

2015-10-06

The solvent accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding, conformational change, and interaction. This fundamental biophysical parameter is elusive in experimental measurement. Our approach to this problem relies on the reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides. This reagent (i) exerts solvent mimicry because its size is comparable to water and (ii) shows scant chemical selectivity because it generates extremely reactive methylene carbene. Methylation gives rise to the EM (extent of modification) signal, which is useful for scrutinizing the conformational change triggered by Ca(2+) binding to calmodulin (CaM). The increased EM observed for the full protein is dominated by the enhanced exposure of hydrophobic area in Ca(2+)-CaM. Fragmentation allowed us to quantify the methylene incorporation at specific sites. Peptide 91-106 reveals a major reorganization around the calcium 151 binding site, resulting in local ordering and a greater exposure of the hydrophobic surface. Additionally, this technique shows a high sensitivity to probe recognition between CaM and melittin (Mel). The large decrease in EM indicates the occlusion of a significant hydrophobic area upon complexation. Protection from labeling reveals a larger involvement of the N-terminal and central regions of CaM in this interaction. Despite its smaller size, Mel's differential exposure can also be quantified. Moreover, MS/MS fragmentation realizes the goal of extending the resolution of labeled sites at the amino acid level. Overall, DZN labeling emerges as a useful footprinting method capable of shedding light on physiological conformational changes and interactions.

The expanded amelogenin polyproline region preferentially binds to apatite versus carbonate and promotes apatite crystal elongation

PubMed Central

Gopinathan, Gokul; Jin, Tianquan; Liu, Min; Li, Steve; Atsawasuwan, Phimon; Galang, Maria-Therese; Allen, Michael; Luan, Xianghong; Diekwisch, Thomas G. H.

2014-01-01

The transition from invertebrate calcium carbonate-based calcite and aragonite exo- and endoskeletons to the calcium phosphate-based vertebrate backbones and jaws composed of microscopic hydroxyapatite crystals is one of the great revolutions in the evolution of terrestrial organisms. To identify potential factors that might have played a role in such a transition, three key domains of the vertebrate tooth enamel protein amelogenin were probed for calcium mineral/protein interactions and their ability to promote calcium phosphate and calcium carbonate crystal growth. Under calcium phosphate crystal growth conditions, only the carboxy-terminus augmented polyproline repeat peptide, but not the N-terminal peptide nor the polyproline repeat peptide alone, promoted the formation of thin and parallel crystallites resembling those of bone and initial enamel. In contrast, under calcium carbonate crystal growth conditions, all three amelogenin-derived polypeptides caused calcium carbonate to form fused crystalline conglomerates. When examined for long-term crystal growth, polyproline repeat peptides of increasing length promoted the growth of shorter calcium carbonate crystals with broader basis, contrary to the positive correlation between polyproline repeat element length and apatite mineralization published earlier. To determine whether the positive correlation between polyproline repeat element length and apatite crystal growth versus the inverse correlation between polyproline repeat length and calcium carbonate crystal growth were related to the binding affinity of the polyproline domain to either apatite or carbonate, a parallel series of calcium carbonate and calcium phosphate/apatite protein binding studies was conducted. These studies demonstrated a remarkable binding affinity between the augmented amelogenin polyproline repeat region and calcium phosphates, and almost no binding to calcium carbonates. In contrast, the amelogenin N-terminus bound to both carbonate and apatite, but preferentially to calcium carbonate. Together, these studies highlight the specific binding affinity of the augmented amelogenin polyproline repeat region to calcium phosphates versus calcium carbonate, and its unique role in the growth of thin apatite crystals as they occur in vertebrate biominerals. Our data suggest that the rise of apatite-based biominerals in vertebrates might have been facilitated by a rapid evolution of specialized polyproline repeat proteins flanked by a charged domain, resulting in apatite crystals with reduced width, increased length, and tailored biomechanical properties. PMID:25426079

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins.

PubMed

Sun, Y; Zhang, J; Kraeft, S K; Auclair, D; Chang, M S; Liu, Y; Sutherland, R; Salgia, R; Griffin, J D; Ferland, L H; Chen, L B

1999-11-19

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

Molecular cloning and analysis of Schizosaccharomyces pombe Reb1p: sequence-specific recognition of two sites in the far upstream rDNA intergenic spacer.

PubMed Central

Zhao, A; Guo, A; Liu, Z; Pape, L

1997-01-01

The coding sequences for a Schizosaccharomyces pombe sequence-specific DNA binding protein, Reb1p, have been cloned. The predicted S. pombe Reb1p is 24-29% identical to mouse TTF-1 (transcription termination factor-1) and Saccharomyces cerevisiae REB1 protein, both of which direct termination of RNA polymerase I catalyzed transcripts. The S.pombe Reb1 cDNA encodes a predicted polypeptide of 504 amino acids with a predicted molecular weight of 58.4 kDa. The S. pombe Reb1p is unusual in that the bipartite DNA binding motif identified originally in S.cerevisiae and Klyveromyces lactis REB1 proteins is uninterrupted and thus S.pombe Reb1p may contain the smallest natural REB1 homologous DNA binding domain. Its genomic coding sequences were shown to be interrupted by two introns. A recombinant histidine-tagged Reb1 protein bearing the rDNA binding domain has two homologous, sequence-specific binding sites in the S. pomber DNA intergenic spacer, located between 289 and 480 nt downstream of the end of the approximately 25S rRNA coding sequences. Each binding site is 13-14 bp downstream of two of the three proposed in vivo termination sites. The core of this 17 bp site, AGGTAAGGGTAATGCAC, is specifically protected by Reb1p in footprinting analysis. PMID:9016645

moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, D.J.; Lidstrom, M.E.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome c/sub L/. In this study, four polypeptides of M/sub r/, 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement ofmore » the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the M/sub r/-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the M/sub r/-20,000 polypeptide, was identified as mature cytochrome c/sub L/, and the product of moxI, the M/sub r/-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the M/sub r/-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the M/sub r/-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the M/sub r/-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the M/sub r/-60,000 MeDH polypeptide. Our data suggest that both the M/sub r/-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.« less

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

PubMed

Shoots, Jenny; Fraccalvieri, Domenico; Franks, Diana G; Denison, Michael S; Hahn, Mark E; Bonati, Laura; Powell, Wade H

2015-06-02

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Probing protein surface with a solvent mimetic carbene coupled to detection by mass spectrometry.

PubMed

Gómez, Gabriela E; Mundo, Mariana R; Craig, Patricio O; Delfino, José M

2012-01-01

Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH(2)). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites. © American Society for Mass Spectrometry, 2011

Phage-displayed specific polypeptide antigens induce significant protective immunity against Trichinella spiralis infection in BALB/c mice.

PubMed

Cui, Jing; Ren, Hui Jun; Liu, Ruo Dan; Wang, Li; Zhang, Zi Fang; Wang, Zhong Quan

2013-02-06

Trichinellosis is a public health problem and is considered an emerging/re-emerging disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects humans, domestic animals and wildlife. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Phage particles are especially ideal vaccine delivery vehicles because they do not interfere with the immune response against the displayed peptide antigens, and, if anything, are more likely to efficiently direct antigen expression to professional antigen-presenting cells. In this study, Tsp10 polypeptide, which was encoded by a cDNA fragment of Trichinella spiralis intestinal infective larvae and was found to bind to normal mouse intestinal cells, was displayed on the surface of T7 phage. Anti-Tsp10 antibodies were able to recognize the native Tsp10 protein mainly localized to the stichosome of T. spiralis. Mice immunized with the recombinant phage T7-Tsp10 showed a 62.8% reduction in adult worms and a 78.6% reduction in muscle larvae following challenge with T. spiralis muscle larvae. Our results demonstrate that the vaccination with Tsp10 polypeptide displayed by T7 phage elicits the Th2-predominant immune responses and produces a significant protection against T. spiralis infection in mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against T. spiralis. Copyright © 2013 Elsevier Ltd. All rights reserved.

The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins.

PubMed

Giga, Y; Ikai, A; Takahashi, K

1987-05-05

The complete amino acid sequence of echinoidin, the proposed name for a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina, has been determined by sequencing the peptides obtained from tryptic, Staphylococcus aureus V8 protease, chymotryptic, and thermolysin digestions. Echinoidin is a multimeric protein (Giga, Y., Sutoh, K., and Ikai, A. (1985) Biochemistry 24, 4461-4467) whose subunit consists of a total of 147 amino acid residues and one carbohydrate chain attached to Ser38. The molecular weight of the polypeptide without carbohydrate was calculated to be 16,671. Each polypeptide chain contains seven half-cystines, and six of them form three disulfide bonds in the single polypeptide chain (Cys3-Cys14, Cys31-Cys141, and Cys116-Cys132), while Cys2 is involved in an interpolypeptide disulfide linkage. From secondary structure prediction by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222) the protein appears to be rich in beta-sheet and beta-turn structures and poor in alpha-helical structure. The sequence of the COOH-terminal half of echinoidin is highly homologous to those of the COOH-terminal carbohydrate recognition portions of rat liver mannose-binding protein and several other hepatic lectins. This COOH-terminal region of echinoidin is also homologous to the central portion of the lectin from the flesh fly Sarcophaga peregrina. Moreover, echinoidin contains an Arg-Gly-Asp sequence which has been proposed to be a basic functional unit in cellular recognition proteins.

Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

PubMed Central

Alegre, Ana Claudia Paiva; Oliveira, Aline Ferreira; Dos Reis Almeida, Fausto Bruno; Roque-Barreira, Maria Cristina; Hanna, Ebert Seixas

2014-01-01

Background Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. Methodology/principal findings The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. Conclusions/Significance Our results showed that the recombinant protein reproduced the biological properties described for the native protein—including binding to laminin in a manner that is dependent on carbohydrate recognition—showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects. PMID:24743161

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.

Lamins in the nuclear interior - life outside the lamina.

PubMed

Naetar, Nana; Ferraioli, Simona; Foisner, Roland

2017-07-01

Nuclear lamins are components of the peripheral lamina that define the mechanical properties of nuclei and tether heterochromatin to the periphery. A-type lamins localize also to the nuclear interior, but the regulation and specific functions of this nucleoplasmic lamin pool are poorly understood. In this Commentary, we summarize known pathways that are potentially involved in the localization and dynamic behavior of intranuclear lamins, including their post-translational modifications and interactions with nucleoplasmic proteins, such as lamina-associated polypeptide 2α (LAP2α; encoded by TMPO ). In addition, new data suggest that lamins in the nuclear interior have an important role in chromatin regulation and gene expression through dynamic binding to both hetero- and euchromatic genomic regions and promoter subdomains, thereby affecting epigenetic pathways and chromatin accessibility. Nucleoplasmic lamins also have a role in spatial chromatin organization and may be involved in mechanosignaling. In view of this newly emerging concept, we propose that the previously reported cellular phenotypes in lamin-linked diseases are, at least in part, rooted in an impaired regulation and/or function of the nucleoplasmic lamin A/C pool. © 2017. Published by The Company of Biologists Ltd.

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

PubMed Central

Siu, Fai Y.; Spanggord, Richard J.; Doudna, Jennifer A.

2007-01-01

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP–receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP–receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP–receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo. PMID:17164479

Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3

PubMed Central

Ferru, Emanuela; Giger, Katie; Pantaleo, Antonella; Campanella, Estela; Grey, Jesse; Ritchie, Ken; Vono, Rosa; Low, Philip S.

2011-01-01

The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses. PMID:21474668

Polypeptide p41 of a Norwalk-Like Virus Is a Nucleic Acid-Independent Nucleoside Triphosphatase

PubMed Central

Pfister, Thomas; Wimmer, Eckard

2001-01-01

Southampton virus (SHV) is a member of the Norwalk-like viruses (NLVs), one of four genera of the family Caliciviridae. The genome of SHV contains three open reading frames (ORFs). ORF 1 encodes a polyprotein that is autocatalytically processed into six proteins, one of which is p41. p41 shares sequence motifs with protein 2C of picornaviruses and superfamily 3 helicases. We have expressed p41 of SHV in bacteria. Purified p41 exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities. The NTPase activity was not stimulated by single-stranded nucleic acids. SHV p41 had no detectable helicase activity. Protein sequence comparison between the consensus sequences of NLV p41 and enterovirus protein 2C revealed regions of high similarity. According to secondary structure prediction, the conserved regions were located within a putative central domain of alpha helices and beta strands. This study reveals for the first time an NTPase activity associated with a calicivirus-encoded protein. Based on enzymatic properties and sequence information, a functional relationship between NLV p41 and enterovirus 2C is discussed in regard to the role of 2C-like proteins in virus replication. PMID:11160659

Immunoglobulin G1 Fc domain motions: implications for Fc engineering

PubMed Central

Frank, Martin; Walker, Ross C.; Lanzilotta, William N.; Prestegard, James H.; Barb, Adam W.

2014-01-01

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G(IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response is tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in Fcγ receptor affinity being modulated through shifts in Fc conformational sampling. Here we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics (MD) investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulphide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population. PMID:24522230

Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae.

PubMed

Eid, Rawan; Boucher, Eric; Gharib, Nada; Khoury, Chamel; Arab, Nagla T T; Murray, Alistair; Young, Paul G; Mandato, Craig A; Greenwood, Michael T

2016-03-01

Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

PubMed

Ott, Wolfgang; Jobst, Markus A; Bauer, Magnus S; Durner, Ellis; Milles, Lukas F; Nash, Michael A; Gaub, Hermann E

2017-06-27

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation.

PubMed

Petitdemange, Rosine; Garanger, Elisabeth; Bataille, Laure; Dieryck, Wilfrid; Bathany, Katell; Garbay, Bertrand; Deming, Timothy J; Lecommandoux, Sébastien

2017-02-13

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media.

Polypeptides having catalase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having catalase activity and 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.

Revisiting structure-property relationship of pH-responsive polymers for drug delivery applications.

PubMed

Bazban-Shotorbani, Salime; Hasani-Sadrabadi, Mohammad Mahdi; Karkhaneh, Akbar; Serpooshan, Vahid; Jacob, Karl I; Moshaverinia, Alireza; Mahmoudi, Morteza

2017-05-10

pH-responsive polymers contain ionic functional groups as pendants in their structure. The total number of charged groups on polymer chains determines the overall response of the system to changes in the external pH. This article reviews various pH-responsive polymers classified as polyacids (e.g., carboxylic acid based polymers, sulfonamides, anionic polysaccharides, and anionic polypeptides) and polybases (e.g., polyamines, pyridine and imidazole containing polymers, cationic polysaccharides, and cationic polypeptides). We correlate the pH variations in the body at the organ level (e.g., gastrointestinal tract and vaginal environment), tissue level (e.g., cancerous and inflamed tissues), and cellular level (e.g., sub-cellular organelles), with the intrinsic properties of pH-responsive polymers. This knowledge could help to select more effective ('smart') polymeric systems based on the biological target. Considering the pH differences in the body, various drug delivery systems can be designed by utilizing smart biopolymeric compounds with the required pH-sensitivity. We also review the pharmaceutical application of pH-responsive polymeric carriers including hydrogels, polymer-drug conjugates, micelles, dendrimers, and polymersomes. © 2016.

Charge transport and intrinsic fluorescence in amyloid-like fibrils

PubMed Central

del Mercato, Loretta Laureana; Pompa, Pier Paolo; Maruccio, Giuseppe; Torre, Antonio Della; Sabella, Stefania; Tamburro, Antonio Mario; Cingolani, Roberto; Rinaldi, Ross

2007-01-01

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications. PMID:17984067

Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly

NASA Astrophysics Data System (ADS)

Mozhdehi, Davoud; Luginbuhl, Kelli M.; Simon, Joseph R.; Dzuricky, Michael; Berger, Rüdiger; Varol, H. Samet; Huang, Fred C.; Buehne, Kristen L.; Mayne, Nicholas R.; Weitzhandler, Isaac; Bonn, Mischa; Parekh, Sapun H.; Chilkoti, Ashutosh

2018-05-01

Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials—fatty-acid-modified elastin-like polypeptides—using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C14 alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity.

PubMed

Magrì, Antonio; La Mendola, Diego; Nicoletti, Vincenzo Giuseppe; Pappalardo, Giuseppe; Rizzarelli, Enrico

2016-09-05

Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu(2+) ions with Ac-PEG-hIAPP(17-29)-NH2 , Ac-rIAPP(17-29)R18H-NH2 , and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu(2+) ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu(2+) ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu(2+) ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2 O2 production attributed to the polypeptide alone. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.

PubMed Central

Gil, M L; Peñalver, M C; Lopez-Ribot, J L; O'Connor, J E; Martinez, J P

1996-01-01

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease. PMID:8945572

The quaternary architecture of RARβ–RXRα heterodimer facilitates domain–domain signal transmission

DOE PAGES

Chandra, Vikas; Wu, Dalei; Li, Sheng; ...

2017-10-11

Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor beta-retinoic X receptor alpha (RAR beta-RXR alpha) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen-deuterium exchange mass spectrometry, mutagenesis and functional studies. The RAR beta ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within theirmore » quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its hetero-dimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs.« less

Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.

PubMed

Ventas, P; Carreira, J; Polo, F

1991-08-01

The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.

The quaternary architecture of RARβ–RXRα heterodimer facilitates domain–domain signal transmission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandra, Vikas; Wu, Dalei; Li, Sheng

Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor beta-retinoic X receptor alpha (RAR beta-RXR alpha) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen-deuterium exchange mass spectrometry, mutagenesis and functional studies. The RAR beta ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within theirmore » quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its hetero-dimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs.« less

Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers

PubMed Central

Feld, Geoffrey K.; Thoren, Katie L.; Kintzer, Alexander F.; Sterling, Harry J.; Tang, Iok I.; Greenberg, Shoshana G.; Williams, Evan R.; Krantz, Bryan A.

2011-01-01

The protein transporter, anthrax lethal toxin, is comprised of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. Following assembly into holotoxin complexes, PA forms an oligomeric channel that unfolds LF and translocates it into the host cell. We report the crystal structure of the core of a lethal toxin complex to 3.1-Å resolution; the structure contains a PA octamer bound to four LF PA-binding domains (LFN). The first α helix and β strand of each LFN unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which we call the α clamp. The α clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA octamer formation, and LF unfolding and translocation. This structure provides insight on the mechanism of translocation-coupled protein unfolding. PMID:21037566

A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

PubMed

Kim, Heejae; Chen, Wilfred

2016-09-20

Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. Copyright © 2016 Elsevier B.V. All rights reserved.