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Sample records for modifies protein complexes

  1. A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC.

    PubMed

    Ng, J; Hart, C M; Morgan, K; Simon, J A

    2000-05-01

    The extra sex combs (ESC) and Enhancer of zeste [E(Z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence that ESC is phosphorylated in vivo and that this modified ESC is preferentially associated in the complex with E(Z). Modified ESC accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. We find that mutations in E(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants that express ESC proteins bearing site-directed mutations that disrupt ESC-E(Z) binding in vitro. These mutant ESC proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that ESC phosphorylation normally occurs after assembly into ESC-E(Z) complexes and that it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression. PMID:10757791

  2. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

    SciTech Connect

    Adams, Michael W.; W. W. Adams, Michael

    2014-01-07

    Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

  3. N-terminal lysines are essential for protein translocation via a modified ERAD system in complex plastids.

    PubMed

    Lau, Julia B; Stork, Simone; Moog, Daniel; Sommer, Maik S; Maier, Uwe G

    2015-05-01

    Nuclear-encoded pre-proteins being imported into complex plastids of red algal origin have to cross up to five membranes. Thereby, transport across the second outermost or periplastidal membrane (PPM) is facilitated by SELMA (symbiont-specific ERAD-like machinery), an endoplasmic reticulum-associated degradation (ERAD)-derived machinery. Core components of SELMA are enzymes involved in ubiquitination (E1-E3), a Cdc48 ATPase complex and Derlin proteins. These components are present in all investigated organisms with four membrane-bound complex plastids of red algal origin, suggesting a ubiquitin-dependent translocation process of substrates mechanistically similar to the process of retro-translocation in ERAD. Even if, according to the current model, translocation via SELMA does not end up in the classical poly-ubiquitination, transient mono-/oligo-ubiquitination of pre-proteins might be required for the mechanism of translocation. We investigated the import mechanism of SELMA and were able to show that protein transport across the PPM depends on lysines in the N-terminal but not in the C-terminal part of pre-proteins. These lysines are predicted to be targets of ubiquitination during the translocation process. As proteins lacking the N-terminal lysines get stuck in the PPM, a 'frozen intermediate' of the translocation process could be envisioned and initially characterized.

  4. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  5. Preparation and functionalization of a visible-light-excited europium complex-modified luminescent protein for cell imaging applications.

    PubMed

    Tian, Lu; Dai, Zhichao; Ye, Zhiqiang; Song, Bo; Yuan, Jingli

    2014-03-01

    Lanthanide complex-based luminescent bioprobes have shown great utility in a variety of time-resolved luminescence bioassays, but these bioprobes often require UV excitation and suffer from problems related to bioaffinity and biocompatibility for in vivo applications. In this work, a new visible-light-excited europium(III) complex with the maximum excitation wavelength over 400 nm, BHHBB-Eu(3+)-BPT {BHHBB: 1,2-bis[4'-(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)-benzyl]-benzene; BPT: 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine}, has been synthesized for the preparation of an artificial luminescent protein that can be used as a visible-light-excited luminescent bioprobe for cell imaging. By encapsulating BHHBB-Eu(3+)-BPT into apoferritin with a simple dissociation-reassembly method, the luminescent protein, Eu@AFt, with a maximum excitation peak at 420 nm and a long luminescence lifetime of 365 μs was fabricated and successfully used for visible-light-excited time-resolved luminescence cell imaging. Moreover, by conjugating a mitochondria-targeting molecule, (5-N-succinimidoxy-5-oxopentyl)-triphenylphosphonium bromide (SPTPP), onto the surface of Eu@AFt, a mitochondria-specifically-tracking luminescent probe, Eu@AFt-SPTPP, was further prepared and used for visible-light-excited confocal luminescence microscopy imaging to visualize the mitochondria of living cells.

  6. Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure

    PubMed Central

    Létoquart, Juliette; van Tran, Nhan; Caroline, Vonny; Aleksandrov, Alexey; Lazar, Noureddine; van Tilbeurgh, Herman; Liger, Dominique; Graille, Marc

    2015-01-01

    Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. PMID:26438534

  7. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  8. Adhesives from modified soy protein

    DOEpatents

    Sun, Susan; Wang, Donghai; Zhong, Zhikai; Yang, Guang

    2008-08-26

    The, present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  9. Suppressor of hairy‐wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi

    PubMed Central

    Carballar‐Lejarazú, R.; Brennock, P.

    2016-01-01

    Abstract DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy‐like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric‐a‐brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood‐fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes. PMID:27110891

  10. Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi.

    PubMed

    Carballar-Lejarazú, R; Brennock, P; James, A A

    2016-08-01

    DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.

  11. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  12. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  13. The manganese stabilizing protein of photosystem II modifies the in vivo deactivation and photoactivation kinetics of the H2O oxidation complex in Synechocystis sp. PCC6803.

    PubMed

    Burnap, R L; Qian, M; Pierce, C

    1996-01-23

    Dark deactivation and photoactivation of H2O-splitting activity were examined in a directed mutant (delta psbO) of Synechocystis sp. PCC6803 lacking the extrinsic manganese-stabilizing protein of the photosystem II (PSII) reaction center complex. Rapid (t1/2 = 10 min) losses of H2O-splitting activity were observed for delta psbO cells kept in the dark, but not for wild-type cells. The loss of H2O-splitting activity by delta psbO cells was suppressed by maintaining the cells under illumination and dark losses were rapidly (t1/2 < 1 min) reversed by light. Photoactivation kinetics of delta psbO and wild-type cells were compared following hydroxylamine extraction of PSII Mn. Photoactivation of delta psbO cells under continuous illumination occurs at an intrinsically faster rate (about 4-fold) than the wild-type. Virtually all of the increase in the rate of photoactivation can be accounted for by a corresponding 4-fold increase in the relative quantum yield of photoactivation as indicated by the yield of photoactivation as a function of flash number. The flash frequency dependence of photoactivation indicates a multi-quantum process in the mutant resembling the wild-type, but with significant increases in yields at all flash frequencies examined. The higher quantum yield of photoactivation in delta psbO cells occurs in the absence of large changes in the kinetics of the rate-limiting dark rearrangement. The results are consistent with increased accessibility (or affinity) and photooxidation of Mn2+ at one or both of the two binding sites involved in the initial stages of the photoactivation mechanism. In the context of previous results, it is proposed that MSP regulates the binding/photooxidation of the second Mn2+ of the photoligation sequence, but not the first.

  14. Scavenger receptor for aldehyde-modified proteins.

    PubMed

    Horiuchi, S; Murakami, M; Takata, K; Morino, Y

    1986-04-15

    This paper describes an unexpectedly broad ligand specificity of a scavenger receptor of sinusoidal liver cells that is responsible for endocytic uptake of formaldehyde-treated bovine serum albumin (f-Alb). Binding of 125I-f-Alb to the isolated cells was effectively inhibited by bovine serum albumin (BSA) modified with aliphatic aldehydes such as glycolaldehye, DL-glyceraldehyde, and propionaldehyde whereas albumin preparations modified by aromatic aldehydes such as pyridoxal, pyridoxal phosphate, salicylaldehyde, and benzaldehyde did not affect this binding process. Binding of 125I-glycolaldehyde-treated BSA to the cells exhibited a saturation kinetics with an apparent Kd = 3.3 micrograms of the ligand/ml. This binding process was inhibited by unlabeled f-Alb as well as by the antibody raised against the f-Alb receptor. Indeed, 125I-glycolaldehyde-treated BSA underwent a rapid plasma clearance (t1/2 approximately 2 min) which was markedly retarded by unlabeled f-Alb. Upon treatment by these aldehydes, other proteins such as ovalbumin, soybean trypsin inhibitor, and hemoglobin were also converted to active ligands for the f-Alb receptor, while no ligand activity was generated with gamma-globulin and RNase A. These results clearly show that the f-Alb receptor, originally described as being specific for f-Alb, exhibits a broad ligand specificity in terms of both aldehydes and proteins and, hence, should be described as a scavenger receptor for aldehyde-modified proteins.

  15. Protein sorting in complex plastids.

    PubMed

    Sheiner, Lilach; Striepen, Boris

    2013-02-01

    Taming a cyanobacterium in a pivitol event of endosymbiosis brought photosynthesis to eukaryotes, and gave rise to the plastids found in glaucophytes, red and green algae, and the descendants of the latter, the plants. Ultrastructural as well as molecular research over the last two decades has demonstrated that plastids have enjoyed surprising lateral mobility across the tree of life. Numerous independent secondary and tertiary endosymbiosis have led to a spread of plastids into a variety of, up to that point, non-photosynthetic lineages. Happily eating and subsequently domesticating one another protists conquered a wide variety of ecological niches. The elaborate evolution of secondary, or complex, plastids is reflected in the numerous membranes that bound them (three or four compared to the two membranes of the primary plastids). Gene transfer to the host nucleus is a hallmark of endosymbiosis and provides centralized cellular control. Here we review how these proteins find their way back into the stroma of the organelle and describe the advances in the understanding of the molecular mechanisms that allow protein translocation across four membranes. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  16. Investigation of modified cottonseed protein adhesives for wood composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several modified cottonseed protein isolates were studied and compared to corresponding soy protein isolates for their adhesive properties when bonded to wood composites. Modifications included treatments with alkali, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and urea. Wood composites...

  17. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  18. The Protein-Sparing Modified Fast Diet

    PubMed Central

    Bakhach, Marwan; Shah, Vaishal; Harwood, Tara; Lappe, Sara; Bhesania, Natalie; Mansoor, Sana; Alkhouri, Naim

    2016-01-01

    Objectives: The protein-sparing modified fast (PSMF) is a rigorous way of rapidly losing a large amount of weight. Although adult studies have shown the PSMF to be effective, data in adolescents are lacking. The aim of this study was to determine the efficacy and safety of the PSMF in severely obese adolescents. Methods: 12 subjects who were evaluated in the Obesity Management Program at the Cleveland Clinic from 2011 to 2014 were included. The subjects were initiated on the PSMF after failing other conventional methods of weight loss. Once the goal weight was achieved, subjects were transitioned to the refeeding phase for weight maintenance. Results: Follow-up was scheduled at 3-month (11 patients) and 6-month (6 patients) intervals. At the 6-month follow-up visit, the average weight loss was 11.19 kg (95% confidence interval = -5.4, -27.8, P = .028), with average of 9.8% from baseline. Fifty percent of subjects had >5% weight loss and 20% had >10% weight loss. Four patients were lost to the follow-up (40%). An improvement was noted in total cholesterol and high-density lipoprotein. Due to a small sample size these results were not statistically significant. Side effects reported by subjects were mild dehydration due to nausea (2 patients), decreased energy (1 patient), and transient labile mood (1 patient). No life-threatening side effects were reported. Conclusion: Our results show that the PSMF diet can be used as an effective and safe method in the outpatient setting for rapid weight loss in adolescents with severe obesity. PMID:27335996

  19. Protein camouflage in cytochrome c-calixarene complexes

    NASA Astrophysics Data System (ADS)

    McGovern, Róise E.; Fernandes, Humberto; Khan, Amir R.; Power, Nicholas P.; Crowley, Peter B.

    2012-07-01

    Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

  20. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  1. Investigation of a protein complex network

    NASA Astrophysics Data System (ADS)

    Mashaghi, A. R.; Ramezanpour, A.; Karimipour, V.

    2004-09-01

    The budding yeast Saccharomyces cerevisiae is the first eukaryote whose genome has been completely sequenced. It is also the first eukaryotic cell whose proteome (the set of all proteins) and interactome (the network of all mutual interactions between proteins) has been analyzed. In this paper we study the structure of the yeast protein complex network in which weighted edges between complexes represent the number of shared proteins. It is found that the network of protein complexes is a small world network with scale free behavior for many of its distributions. However we find that there are no strong correlations between the weights and degrees of neighboring complexes. To reveal non-random features of the network we also compare it with a null model in which the complexes randomly select their proteins. Finally we propose a simple evolutionary model based on duplication and divergence of proteins.

  2. SERF protein is a direct modifier of amyloid fiber assembly.

    PubMed

    Falsone, S Fabio; Meyer, N Helge; Schrank, Evelyne; Leitinger, Gerd; Pham, Chi L L; Fodero-Tavoletti, Michelle T; Holmberg, Mats; Dulle, Martin; Scicluna, Benjamin; Gesslbauer, Bernd; Rückert, Hanna-Marie; Wagner, Gabriel E; Merle, David A; Nollen, Ellen A; Kungl, Andreas J; Hill, Andrew F; Cappai, Roberto; Zangger, Klaus

    2012-08-30

    The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway.

  3. SERF Protein Is a Direct Modifier of Amyloid Fiber Assembly

    PubMed Central

    Falsone, S. Fabio; Meyer, N. Helge; Schrank, Evelyne; Leitinger, Gerd; Pham, Chi L.L.; Fodero-Tavoletti, Michelle T.; Holmberg, Mats; Dulle, Martin; Scicluna, Benjamin; Gesslbauer, Bernd; Rückert, Hanna-Marie; Wagner, Gabriel E.; Merle, David A.; Nollen, Ellen A.; Kungl, Andreas J.; Hill, Andrew F.; Cappai, Roberto; Zangger, Klaus

    2012-01-01

    Summary The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway. PMID:22854022

  4. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  5. Enhanced protein internalization and efficient endosomal escape using polyampholyte-modified liposomes and freeze concentration.

    PubMed

    Ahmed, Sana; Fujita, Satoshi; Matsumura, Kazuaki

    2016-09-21

    Here we show a new strategy for efficient freeze concentration-mediated cytoplasmic delivery of proteins, obtained via the endosomal escape property of polyampholyte-modified liposomes. The freeze concentration method successfully induces the efficient internalization of proteins simply by freezing cells with protein and nanocarrier complexes. However, the mechanism of protein internalization remains unclear. Here, we designed a novel protein delivery carrier by modifying liposomes through incorporating hydrophobic polyampholytes therein. These complexes were characterized for particle size, encapsulation efficiency, and cytotoxicity. Flow cytometry and microscopic analysis showed that the adsorption and internalization of protein-loaded polyampholyte-modified liposomes after freezing were enhanced compared with that observed in unfrozen complexes. Inhibition studies demonstrated that the internalization mechanism differs between unmodified and polyampholyte-modified liposomes. Furthermore, polyampholyte-modified liposomes exhibited high efficacy in facilitating endosomal escape to enhance protein delivery to the cytoplasm with low toxicity. These results strongly suggest that the freeze concentration-based strategy could be widely utilised for efficient cargo delivery into the cytoplasm in vitro not only in cancer treatment but also for gene therapy as well. PMID:27439774

  6. Rho-modifying bacterial protein toxins.

    PubMed

    Aktories, Klaus

    2015-12-01

    Rho proteins are targets of numerous bacterial protein toxins, which manipulate the GTP-binding proteins by covalent modifications, including ADP ribosylation, glycosylation, adenylylation, proteolytic cleavage and deamidation. Bacterial toxins are important virulence factors but are also potent and efficient pharmacological tools to study the physiological functions of their eukaryotic targets. Recent studies indicate that amazing variations exist in the molecular mechanisms by which toxins attack Rho proteins, which are discussed here.

  7. Melatonin modifies the rhythm of protein synthesis.

    PubMed

    Brodsky, V Y; Dubovaya, N D; Zvezdina, T K; Fateeva, V I; Mal'chenko, L A

    2010-07-01

    Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.

  8. Protein import into complex plastids: Cellular organization of higher complexity.

    PubMed

    Maier, Uwe G; Zauner, Stefan; Hempel, Franziska

    2015-01-01

    Many protists with high ecological and medical relevance harbor plastids surrounded by four membranes. Thus, nucleus-encoded proteins of these complex plastids have to traverse these barriers. Here we report on the identification of the protein translocators located in two of the plastid surrounding membranes and present recent findings on the mechanisms of protein import into the plastids of diatoms.

  9. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  10. Co-translational assembly of protein complexes.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2015-12-01

    The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

  11. Complex Reconstitution from Individual Protein Modules.

    PubMed

    Basquin, Jérôme; Taschner, Michael; Lorentzen, Esben

    2016-01-01

    Cellular function relies on protein complexes that work as nano-machines. The structure and function of protein complexes is an outcome of the specific combination of protein subunits, or modules, within the complex. A major focus of molecular biology is thus to understand how protein subunits assemble to form complexes with distinct biological function. To this end, in vitro reconstitution of complexes from individual subunits to study their assembly, structure and activity is of central importance. With purified individual subunits and sub-modules at hand one can systematically dissect the hierarchical assembly of larger complexes using direct protein-protein interaction assays. Furthermore, activity assays can be carried out with individual subunits or smaller sub-complexes and compared to those of the fully assembled complex to precisely map functional sites and provide a molecular basis for in vivo observations. In this chapter we review methods for protein complex assembly from individual subunits and provide examples of advantages and potential pitfalls to this approach. PMID:27165333

  12. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  13. Genetically modified proteins: functional improvement and chimeragenesis

    PubMed Central

    Balabanova, Larissa; Golotin, Vasily; Podvolotskaya, Anna; Rasskazov, Valery

    2015-01-01

    This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit. PMID:26211369

  14. 3D complex: a structural classification of protein complexes.

    PubMed

    Levy, Emmanuel D; Pereira-Leal, Jose B; Chothia, Cyrus; Teichmann, Sarah A

    2006-11-17

    Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

  15. Preparation and evaluation of tara-modified proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quebracho, a vegetable tannin, can be used to modify gelatin to produce a product that has been applied effectively as a filler in leather processing, as described in our previous report. In this ongoing study, another vegetable tannin tara is examined for its possible application in protein modifi...

  16. Protein complexes that control renal epithelial polarity

    PubMed Central

    Pieczynski, Jay

    2011-01-01

    Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions. PMID:21228104

  17. [Carbonyl stress and oxidatively modified proteins in chronic renal failure].

    PubMed

    Bargnoux, A-S; Morena, M; Badiou, S; Dupuy, A-M; Canaud, B; Cristol, J-P

    2009-01-01

    Oxidative stress is commonly observed in chronic renal failure patients resulting from an unbalance between overproduction of reactive oxygen species and impairement of defense mechanisms. Proteins appear as potential targets of uremia-induced oxidative stress and may undergo qualitative modifications. Proteins could be directly modified by reactive oxygen species which leads to amino acid oxydation and cross-linking. Proteins could be indirectly modified by reactive carbonyl compounds produced by glycoxidation and lipo-peroxidation. The resulting post-traductional modifications are known as carbonyl stress. In addition, thiols could be oxidized or could react with homocystein leading to homocysteinylation. Finally, tyrosin could be oxidized by myeloperoxidase leading to advanced oxidative protein products (AOPP). Oxidatively modified proteins are increased in chronic renal failure patients and may contribute to exacerbate the oxidative stress/inflammation syndrome. They have been involved in long term complications of uremia such as amyloidosis and accelerated atherosclerosis. PMID:19297289

  18. An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

    PubMed Central

    J Gingell, Joseph; Simms, John; Barwell, James; Poyner, David R; Watkins, Harriet A; Pioszak, Augen A; Sexton, Patrick M; Hay, Debbie L

    2016-01-01

    G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor

  19. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

    Greco, Todd M.; Miteva, Yana; Conlon, Frank L.; Cristea, Ileana M.

    2013-01-01

    Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes. PMID:22956100

  20. The Claudin Megatrachea Protein Complex*

    PubMed Central

    Jaspers, Martin H. J.; Nolde, Kai; Behr, Matthias; Joo, Seol-hee; Plessmann, Uwe; Nikolov, Miroslav; Urlaub, Henning; Schuh, Reinhard

    2012-01-01

    Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates. PMID:22930751

  1. A Modified Tactile Brush Algorithm for Complex Touch Gestures

    SciTech Connect

    Ragan, Eric

    2015-01-01

    Several researchers have investigated phantom tactile sensation (i.e., the perception of a nonexistent actuator between two real actuators) and apparent tactile motion (i.e., the perception of a moving actuator due to time delays between onsets of multiple actuations). Prior work has focused primarily on determining appropriate Durations of Stimulation (DOS) and Stimulus Onset Asynchronies (SOA) for simple touch gestures, such as a single finger stroke. To expand upon this knowledge, we investigated complex touch gestures involving multiple, simultaneous points of contact, such as a whole hand touching the arm. To implement complex touch gestures, we modified the Tactile Brush algorithm to support rectangular areas of tactile stimulation.

  2. Interaction proteomics of synapse protein complexes

    PubMed Central

    Klemmer, Patricia; Smit, August B.

    2010-01-01

    The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome. PMID:20361179

  3. Role of Histone-Modifying Enzymes and Their Complexes in Regulation of Chromatin Biology.

    PubMed

    DesJarlais, Renee; Tummino, Peter J

    2016-03-22

    In 1964, Alfrey and colleagues proposed that acetylation and methylation of histones may regulate RNA synthesis and described "the possibility that relatively minor modifications of histone structure, taking place on the intact protein molecule, offer a means of switching-on or off RNA synthesis at different loci along the chromosome" [Allfrey, V., Faulkner, R., and Mirsky, A. (1964) Proc. Natl. Acad. Sci. U.S.A. 51, 786]. Fifty years later, this prescient description provides a simple but conceptually accurate model for the biological role of histone post-translational modifications (PTMs). The basic unit of chromosomes is the nucleosome, with double-stranded DNA wrapped around a histone protein oligomer. The "tails" of histone proteins are post-translationally modified, which alters the physical properties of nucleosomes in a manner that impacts gene accessibility for transcription and replication. Enzymes that catalyze the addition and removal of histone PTMs, histone-modifying enzymes (HMEs), are present in large protein complexes, with DNA-binding proteins, ATP-dependent chromatin remodeling enzymes, and epigenetic reader proteins that bind to post-translationally modified histone residues [Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K., and Schapira, M. (2012) Nat. Rev. Drug Discovery 11, 384-400]. The activity of HME complexes is coordinated with that of other chromatin-associated complexes that, together, regulate gene transcription, DNA repair, and DNA replication. In this context, the enzymes that catalyze addition and removal of histone PTMs are an essential component of the highly regulated mechanism for accessing compacted DNA. To fully understand the function of HMEs, the structure of nucleosomes, their natural substrate, will be described. Each major class of HMEs subsequently will be discussed with regard to its biochemistry, enzymatic mechanism, and biological function in the context of a prototypical HME complex.

  4. Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes.

    PubMed

    Guelorget, Amandine; Golinelli-Pimpaneau, Béatrice

    2011-03-01

    Posttranscriptional chemical modifications of RNA are maturation steps necessary for their correct functioning in translation during protein synthesis. Various structures of RNA-modifying enzymes complexed with RNA fragments or full-length tRNA have been obtained, mimicking several stages along the catalytic cycle such as initial RNA binding, covalent intermediate formation, or RNA-product binding. We summarize here the strategies that have been used to trap and crystallize these stable complexes. Absence of the cosubstrate transferring the chemical group leads to the Michaelis complex, whereas use of a cosubstrate analog to a ternary complex. 5-fluoro-pyrimidine-containing mini RNAs have been used as a general means to trap RNA m(5)U methyltransferase covalent complexes and RNA product/pseudouridine synthase complexes. Altogether, these structures have brought key information about enzyme/RNA recognition and highlighted the details of several catalytic steps of the reactions.

  5. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

    PubMed

    Kapphahn, Rebecca J; Giwa, Babatomiwa M; Berg, Kristin M; Roehrich, Heidi; Feng, Xiao; Olsen, Timothy W; Ferrington, Deborah A

    2006-07-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification. PMID:16530755

  6. Effects of Chemically Modified Messenger RNA on Protein Expression.

    PubMed

    Li, Bin; Luo, Xiao; Dong, Yizhou

    2016-03-16

    Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs. PMID:26906521

  7. Complex coacervation of supercharged proteins with polyelectrolytes.

    PubMed

    Obermeyer, Allie C; Mills, Carolyn E; Dong, Xue-Hui; Flores, Romeo J; Olsen, Bradley D

    2016-04-21

    Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

  8. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  9. Optimization of the electrostatic interactions in protein-protein complexes

    NASA Astrophysics Data System (ADS)

    Alexov, Emil; Brock, Kelly; Kundrotas, Petras

    2007-03-01

    Electrostatic energy is one of the driving forces of protein-protein association. Understanding the role of the energy components on the energetics of protein-protein association will help us in engineering protein-protein interactions and could lead to development of scoring functions that can rank alternative models and decoys. Here we investigate whether the components of the electrostatic energy of protein-protein complexes is optimized in respect to random distribution of the charged residues. We report a clear tendency that coulombic electrostatic interactions are optimized, while the reaction field energy is inversely optimized. It was found that the maximum of the coulombic energy Z-score is shifted 3 units away from the origin and the maximum of the reaction field energy by 2 units. Such a large shift of the Z-score of both coulombic and reaction field energies indicates that wild-type protein-protein interactions are in most cases optimized in terms of coulombic interactions while compromising reaction field energy. Based on these finding a scoring function was developed as a linear combination of the Z-score of the coulombic interactions minus Z-score of the reaction field energy. The scoring function was tested against the decoy sets and it was shown that in majority of the cases we can identify the wild-type complex among hundreds of decoys.

  10. Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

    PubMed Central

    Arur, Swathi; Schedl, Tim

    2014-01-01

    Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

  11. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  12. Measuring the functional sequence complexity of proteins

    PubMed Central

    Durston, Kirk K; Chiu, David KY; Abel, David L; Trevors, Jack T

    2007-01-01

    Background Abel and Trevors have delineated three aspects of sequence complexity, Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC) observed in biosequences such as proteins. In this paper, we provide a method to measure functional sequence complexity. Methods and Results We have extended Shannon uncertainty by incorporating the data variable with a functionality variable. The resulting measured unit, which we call Functional bit (Fit), is calculated from the sequence data jointly with the defined functionality variable. To demonstrate the relevance to functional bioinformatics, a method to measure functional sequence complexity was developed and applied to 35 protein families. Considerations were made in determining how the measure can be used to correlate functionality when relating to the whole molecule and sub-molecule. In the experiment, we show that when the proposed measure is applied to the aligned protein sequences of ubiquitin, 6 of the 7 highest value sites correlate with the binding domain. Conclusion For future extensions, measures of functional bioinformatics may provide a means to evaluate potential evolving pathways from effects such as mutations, as well as analyzing the internal structural and functional relationships within the 3-D structure of proteins. PMID:18062814

  13. Complexation thermodynamics of modified cyclodextrins: extended cavities and distorted structures.

    PubMed

    Schönbeck, Christian; Westh, Peter; Holm, René

    2014-08-28

    Inclusion complexes between two bile salts and a range of differently methylated β-cyclodextrins were studied in an attempt to rationalize the complexation thermodynamics of modified cyclodextrins. Calorimetric titrations at a range of temperatures provided precise values of the enthalpies (ΔH°), entropies (ΔS°), and heat capacities (ΔCp) of complexation, while molecular dynamics simulations assisted the interpretation of the obtained thermodynamic parameters. As previously observed for several types of modified cyclodextrins, the substituents at the rims of the cyclodextrin induced large changes in ΔH° and ΔS°, but due to enthalpy-entropy compensation the changes in Gibbs free energy, and the binding constant, were much smaller. For the methylated β-cyclodextrins, the substituent-induced increments in ΔH° and ΔS° were nonmonotonic with an initial strong increase in both ΔH° and ΔS° and then a strong decrease when the degree of substitution exceeded some threshold. Exactly the same trend was observed for ΔCp. The dehydration of nonpolar surface, as quantified by the simulations, can to a large extent explain the variation in the thermodynamic parameters. The methyl substituents form additional hydrophobic contacts with the bile salt, but at high degrees of methylation they also cause significant distortion of the otherwise circular cyclodextrin structure. These two opposing contributions to the dehydration are the major causes for the observed variations in the thermodynamic functions. The structural effects are not expected to be specific for methylated cyclodextrins but should be observed for most modified cyclodextrins. An even more general conclusion is that variations in the extent of hydration are an important underlying reason for the commonly observed phenomenon termed enthalpy-entropy compensation and also for the less frequent reports of entropy convergence around 110 °C.

  14. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  15. Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    PubMed Central

    Esteve, Juan M.; Villarroya, Magda; Aguado, Carmen; Enríquez, J. Antonio; Knecht, Erwin; Armengod, M.-Eugenia

    2015-01-01

    GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function. PMID:26642043

  16. A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios.

    PubMed

    Blatt, Andreas; Bauch, Matthias E; Pörschke, Yvonne; Lohr, Martin

    2015-05-01

    Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and β-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-β-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

  17. Complex micropatterning of proteins within microfluidic channels.

    PubMed

    Kim, Miju; Doh, Junsang

    2014-01-01

    Microfluidic channels containing protein micropatterned surfaces are useful in many bioanalytical and biological applications. In this study, we developed a new method to integrate microfluidics and protein micropatterning by attaching poly(dimethylsiloxane) (PDMS) microfluidic channels to bio-friendly photoresist films via poly(dopamine) (PDA) adhesive. A bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP) was synthesized and used. By performing microscope projection photolithography (MPP) to the PDMP thin films within PDMS microchannels, complex micropatterns of proteins were successfully generated within microfluidic channels. PMID:25570075

  18. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-01

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  19. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  20. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

    Mass spectrometry, an analytical technique that measures the mass-to-charge ratio of ionized atoms or molecules, dates back more than 100 years, and has both qualitative and quantitative uses for determining chemical and structural information. Quantitative proteomic mass spectrometry on biological samples focuses on identifying the proteins present in the samples, and establishing the relative abundances of those proteins. Such protein inventories create the opportunity to discover novel biomarkers and disease targets. We have previously introduced a normalized, label-free method for quantification of protein abundances under a shotgun proteomics platform (Griffin et al., 2010). The introduction of this method for quantifying and comparing protein levels leads naturally to the issue of modeling protein abundances in individual samples. We here report that protein abundance levels from two recent proteomics experiments conducted by the authors can be adequately represented by Sichel distributions. Mathematically, Sichel distributions are mixtures of Poisson distributions with a rather complex mixing distribution, and have been previously and successfully applied to linguistics and species abundance data. The Sichel model can provide a direct measure of the heterogeneity of protein abundances, and can reveal protein abundance differences that simpler models fail to show. PMID:23360617

  1. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

  2. Identification of Proteins that Modify Cataract of the Eye Lens

    PubMed Central

    Hoehenwarter, Wolfgang; Tang, Yajun; Ackermann, Renate; Pleissner, Klaus-Peter; Schmid, Monika; Stein, Robert; Zimny-Arndt, Ursula; Kumar, Nalin M.; Jungblut, Peter R.

    2010-01-01

    The occurrence of a nuclear cataract in the eye lens due to disruption of theα3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and post-translational modifications occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29 and syntaxin binding protein 6 in the eye lens. DNA polymorphisms resulting in non-conservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1 and possibly gamma N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat shock proteins have a major role for influencing cataract formation in humans. PMID:19003866

  3. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  4. Isolation of proteins and protein complexes by immunoprecipitation.

    PubMed

    Kaboord, Barbara; Perr, Maria

    2008-01-01

    Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.

  5. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  6. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  7. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  8. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  9. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under...

  10. Preparation of Modified Films with Protein from Grouper Fish

    PubMed Central

    Tecante, A.; Granados-Navarrete, S.; Martínez-García, C.

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials. PMID:27597950

  11. Preparation of Modified Films with Protein from Grouper Fish

    PubMed Central

    Tecante, A.; Granados-Navarrete, S.; Martínez-García, C.

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials.

  12. Preparation of Modified Films with Protein from Grouper Fish.

    PubMed

    Valdivia-López, M A; Tecante, A; Granados-Navarrete, S; Martínez-García, C

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials.

  13. Preparation of Modified Films with Protein from Grouper Fish.

    PubMed

    Valdivia-López, M A; Tecante, A; Granados-Navarrete, S; Martínez-García, C

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials. PMID:27597950

  14. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    PubMed

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  15. Tuning of protein-surfactant interaction to modify the resultant structure.

    PubMed

    Mehan, Sumit; Aswal, Vinod K; Kohlbrecher, Joachim

    2015-09-01

    Small-angle neutron scattering and dynamic light scattering studies have been carried out to examine the interaction of bovine serum albumin (BSA) protein with different surfactants under varying solution conditions. We show that the interaction of anionic BSA protein (pH7) with surfactant and the resultant structure are strongly modified by the charge head group of the surfactant, ionic strength of the solution, and mixed surfactants. The protein-surfactant interaction is maximum when two components are oppositely charged, followed by components being similarly charged through the site-specific binding, and no interaction in the case of a nonionic surfactant. This interaction of protein with ionic surfactants is characterized by the fractal structure representing a bead-necklace structure of micellelike clusters adsorbed along the unfolded protein chain. The interaction is enhanced with ionic strength only in the case of site-specific binding of an anionic surfactant with an anionic protein, whereas it is almost unchanged for other complexes of cationic and nonionic surfactants with anionic proteins. Interestingly, the interaction of BSA protein with ionic surfactants is significantly suppressed in the presence of nonionic surfactant. These results with mixed surfactants thus can be used to fold back the unfolded protein as well as to prevent surfactant-induced protein unfolding. For different solution conditions, the results are interpreted in terms of a change in fractal dimension, the overall size of the protein-surfactant complex, and the number of micelles attached to the protein. The interplay of electrostatic and hydrophobic interactions is found to govern the resultant structure of complexes.

  16. Modified Scaled Hierarchical Equation of Motion Approach for the Study of Quantum Coherence in Photosynthetic Complexes

    SciTech Connect

    Zhu, J.; Kais, S.; Rebentrost, P.; Aspuru-Guzik, Alan

    2011-02-17

    We present a detailed theoretical study of the transfer of electronic excitation energy through the Fenna-Matthews-Olson (FMO) pigment-protein complex, using the newly developed modified scaled hierarchical approach (Shi, Q.; et al. J. Chem. Phys.2009, 130, 084105). We show that this approach is computationally more efficient than the original hierarchical approach. The modified approach reduces the truncation levels of the auxiliary density operators and the correlation function. We provide a systematic study of how the number of auxiliary density operators and the higher-order correlation functions affect the exciton dynamics. The time scales of the coherent beating are consistent with experimental observations. Furthermore, our theoretical results exhibit population beating at physiological temperature. Additionally, the method does not require a low-temperature correction to obtain the correct thermal equilibrium at long times.

  17. Radiation damage to DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Spotheim-Maurizot, M.; Davídková, M.

    2011-01-01

    We review here the advances in understanding the effects of ionizing radiations on DNA, proteins and their complexes, resulting from the collaboration of the authors' teams. It concerns the preponderant indirect effect of low LET ionizing radiations, thus the attack of the macromolecules in aqueous solution by the most aggressive product of water radiolysis, the hydroxyl radical. A model of simulation of the reaction of these radicals with the macromolecules (called RADACK) was developed and was used for calculating the probabilities of damage of each constituent of DNA or proteins (nucleotide or amino-acid). The calculations allowed to draw conclusions from electrophoresis, mutagenesis, spectroscopic (fluorescence, circular dichroïsm) and mass spectrometry experiments. Thus we have shown that the extent and location of the lesions are strongly dependent on the 3D structure of the macromolecules, which in turns is modulated by their sequence and by the binding of some ligands. Molecular dynamics simulation completed our studies in showing the consequences of each lesion on the stability and structure of the proteins and their complexes with DNA.

  18. Modeling and fitting protein-protein complexes to predict change of binding energy

    PubMed Central

    Dourado, Daniel F.A.R.; Flores, Samuel Coulbourn

    2016-01-01

    It is possible to accurately and economically predict change in protein-protein interaction energy upon mutation (ΔΔG), when a high-resolution structure of the complex is available. This is of growing usefulness for design of high-affinity or otherwise modified binding proteins for therapeutic, diagnostic, industrial, and basic science applications. Recently the field has begun to pursue ΔΔG prediction for homology modeled complexes, but so far this has worked mostly for cases of high sequence identity. If the interacting proteins have been crystallized in free (uncomplexed) form, in a majority of cases it is possible to find a structurally similar complex which can be used as the basis for template-based modeling. We describe how to use MMB to create such models, and then use them to predict ΔΔG, using a dataset consisting of free target structures, co-crystallized template complexes with sequence identify with respect to the targets as low as 44%, and experimental ΔΔG measurements. We obtain similar results by fitting to a low-resolution Cryo-EM density map. Results suggest that other structural constraints may lead to a similar outcome, making the method even more broadly applicable. PMID:27173910

  19. Interaction of receptor-activity-modifying protein1 with tubulin.

    PubMed

    Kunz, Thomas H; Mueller-Steiner, Sarah; Schwerdtfeger, Kerstin; Kleinert, Peter; Troxler, Heinz; Kelm, Jens M; Ittner, Lars M; Fischer, Jan A; Born, Walter

    2007-08-01

    Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors. PMID:17493758

  20. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H.

    2015-01-01

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. PMID:25564609

  1. Antitumor cell-complex vaccines employing genetically modified tumor cells and fibroblasts.

    PubMed

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Diaz, Ana; Algás, Rosa; Aliño, Salvador F

    2014-02-19

    The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok) and a low producer (p2F). Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP) IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01). When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05). Significant survival (40%) was only observed in the groups vaccinated with free transfected B16 cells.

  2. Antitumor Cell-Complex Vaccines Employing Genetically Modified Tumor Cells and Fibroblasts

    PubMed Central

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Diaz, Ana; Algás, Rosa; Aliño, Salvador F.

    2014-01-01

    The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok) and a low producer (p2F). Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP) IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01). When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05). Significant survival (40%) was only observed in the groups vaccinated with free transfected B16 cells. PMID:24556729

  3. Prioritizing protein complexes implicated in human diseases by network optimization

    PubMed Central

    2014-01-01

    Background The detection of associations between protein complexes and human inherited diseases is of great importance in understanding mechanisms of diseases. Dysfunctions of a protein complex are usually defined by its member disturbance and consequently result in certain diseases. Although individual disease proteins have been widely predicted, computational methods are still absent for systematically investigating disease-related protein complexes. Results We propose a method, MAXCOM, for the prioritization of candidate protein complexes. MAXCOM performs a maximum information flow algorithm to optimize relationships between a query disease and candidate protein complexes through a heterogeneous network that is constructed by combining protein-protein interactions and disease phenotypic similarities. Cross-validation experiments on 539 protein complexes show that MAXCOM can rank 382 (70.87%) protein complexes at the top against protein complexes constructed at random. Permutation experiments further confirm that MAXCOM is robust to the network structure and parameters involved. We further analyze protein complexes ranked among top ten for breast cancer and demonstrate that the SWI/SNF complex is potentially associated with breast cancer. Conclusions MAXCOM is an effective method for the discovery of disease-related protein complexes based on network optimization. The high performance and robustness of this approach can facilitate not only pathologic studies of diseases, but also the design of drugs targeting on multiple proteins. PMID:24565064

  4. Intensity of anxiety is modified via complex integrative stress circuitries.

    PubMed

    Smith, Justin P; Prince, Melissa A; Achua, Justin K; Robertson, James M; Anderson, Raymond T; Ronan, Patrick J; Summers, Cliff H

    2016-01-01

    Escalation of anxious behavior while environmentally and socially relevant contextual events amplify the intensity of emotional response produces a testable gradient of anxiety shaped by integrative circuitries. Apprehension of the Stress-Alternatives Model apparatus (SAM) oval open field (OF) is measured by the active latency to escape, and is delayed by unfamiliarity with the passageway. Familiar OF escape is the least anxious behavior along the continuum, which can be reduced by anxiolytics such as icv neuropeptide S (NPS). Social aggression increases anxiousness in the SAM, reducing the number of mice willing to escape by 50%. The apprehension accompanying escape during social aggression is diminished by anxiolytics, such as exercise and corticotropin releasing-factor receptor 1 (CRF1) antagonism, but exacerbated by anxiogenic treatment, like antagonism of α2-adrenoreceptors. What is more, the anxiolytic CRF1 and anxiogenic α2-adrenoreceptor antagonists also modify behavioral phenotypes, with CRF1 antagonism allowing escape by previously submissive animals, and α2-adrenoreceptor antagonism hindering escape in mice that previously engaged in it. Gene expression of NPS and brain-derived neurotrophic factor (BDNF) in the central amygdala (CeA), as well as corticosterone secretion, increased concomitantly with the escalating anxious content of the mouse-specific anxiety continuum. The general trend of CeA NPS and BDNF expression suggested that NPS production was promoted by increasing anxiousness, and that BDNF synthesis was associated with learning about ever-more anxious conditions. The intensity gradient for anxious behavior resulting from varying contextual conditions may yield an improved conceptualization of the complexity of mechanisms producing the natural continuum of human anxious conditions, and potential therapies that arise therefrom. PMID:26555428

  5. Complex coacervate core micelles with a lysozyme-modified corona.

    PubMed

    Danial, Maarten; Klok, Harm-Anton; Norde, Willem; Stuart, Martien A Cohen

    2007-07-17

    This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.

  6. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking.

    PubMed

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  7. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking

    PubMed Central

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  8. Probing nanoparticle effect in protein-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  9. [Protein assay by the modified Dumas method applied to preparations of plasma proteins].

    PubMed

    Blondel, P; Vian, L

    1993-01-01

    Quantify protein according Pharmacopoeia method, based on Kjeldahl method, needs a long time to do. The development of an automaton which used the modified Dumas method divide the analysis time by 15 (6 minutes versus over 90 minutes). The results show no statistical differences between official method and this one. PMID:8154798

  10. Polyethyleneimine-modified graphene oxide nanocomposites for effective protein functionalization

    NASA Astrophysics Data System (ADS)

    Weng, Yejing; Jiang, Bo; Yang, Kaiguang; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2015-08-01

    A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions.A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions. Electronic supplementary information (ESI) available: Cell viability assay, enrichment of standard glycoprotein, pretreatment and analysis of real

  11. Engineering of complex protein sialylation in plants.

    PubMed

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-08-23

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  12. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  13. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.

  14. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks.

  15. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    NASA Astrophysics Data System (ADS)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  16. A complex-centric view of protein network evolution.

    PubMed

    Yosef, Nir; Kupiec, Martin; Ruppin, Eytan; Sharan, Roded

    2009-07-01

    The recent availability of protein-protein interaction networks for several species makes it possible to study protein complexes in an evolutionary context. In this article, we present a novel network-based framework for reconstructing the evolutionary history of protein complexes. Our analysis is based on generalizing evolutionary measures for single proteins to the level of whole subnetworks, comprehensively considering a broad set of computationally derived complexes and accounting for both sequence and interaction changes. Specifically, we compute sets of orthologous complexes across species, and use these to derive evolutionary rate and age measures for protein complexes. We observe significant correlations between the evolutionary properties of a complex and those of its member proteins, suggesting that protein complexes form early in evolution and evolve as coherent units. Additionally, our approach enables us to directly quantify the extent to which gene duplication has played a role in the evolution of complexes. We find that about one quarter of the sets of orthologous complexes have originated from evolutionary cores of homodimers that underwent duplication and divergence, testifying to the important role of gene duplication in protein complex evolution. PMID:19465379

  17. The SWIRM domain: a conserved module found in chromosomal proteins points to novel chromatin-modifying activities

    PubMed Central

    Aravind, L; Iyer, Lakshminarayan M

    2002-01-01

    Background Eukaryotic chromosomal components, especially histones, are subject to a wide array of covalent modifications and catalytic reorganization. These modifications have an important role in the regulation of chromatin structure and are mediated by large multisubunit complexes that contain modular proteins with several conserved catalytic and noncatalytic adaptor domains. Results Using computational sequence-profile analysis methods, we identified a previously uncharacterized, predicted α-helical domain of about 85 residues in chromosomal proteins such as Swi3p, Rsc8p, Moira and several other uncharacterized proteins. This module, termed the SWIRM domain, is predicted to mediate specific protein-protein interactions in the assembly of chromatin-protein complexes. In one group of proteins, which are highly conserved throughout the crown-group eukaryotes, the SWIRM domain is linked to a catalytic domain related to the monoamine and polyamine oxidases. Another human protein has the SWIRM domain linked to a JAB domain that is involved in protein degradation through the ubiquitin pathway. Conclusions Identification of the SWIRM domain could help in directed experimental analysis of specific interactions in chromosomal proteins. We predict that the proteins in which it is combined with an amino-oxidase domain define a novel class of chromatin-modifying enzymes, which are likely to oxidize either the amino group of basic residues in histones and other chromosomal proteins or the polyamines in chromatin, and thereby alter the charge distribution. Other forms, such as KIAA1915, may link chromatin modification to ubiquitin-dependent protein degradation. PMID:12186646

  18. Antifreeze proteins modify the freezing process in planta.

    PubMed

    Griffith, Marilyn; Lumb, Chelsey; Wiseman, Steven B; Wisniewski, Michael; Johnson, Robert W; Marangoni, Alejandro G

    2005-05-01

    During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.

  19. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  20. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  1. Carbohydrate-protein interactions investigated on plastic chips statically coated with hydrophobically modified hydroxyethylcellulose.

    PubMed

    Dang, Fuquan; Maeda, Eiki; Osafune, Tomo; Nakajima, Kazuki; Kakehi, Kazuaki; Ishikawa, Mitsuru; Baba, Yoshinobu

    2009-12-15

    We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from alpha1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC.

  2. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    NASA Astrophysics Data System (ADS)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  3. Proteins modified by the lipid peroxidation aldehyde DODE in MCF7 breast cancer cells

    PubMed Central

    Slade, Peter G.; Williams, Michelle V.; Brahmbatt, Viral; Dash, Ajit; Wishnok, John S.; Tannenbaum, Steven R.

    2010-01-01

    The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE) which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were use to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by 2D-LC-MS/MS. Second, cells were treated with native 13-HPODE. Protein-carbonyls were biotinylated with an aldehyde reactive probe (ARP) and modified proteins enriched by neutravidin affinity and identified by 2D-LC-MS/MS. Third, using a newly developed DODE antibody, DODE modified proteins were located by 2D-SDS-PAGE and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress. PMID:20131800

  4. Mesoscale assembly of chemically modified graphene into complex cellular networks

    NASA Astrophysics Data System (ADS)

    Barg, Suelen; Perez, Felipe Macul; Ni, Na; Do Vale Pereira, Paula; Maher, Robert C.; Garcia-Tuñon, Esther; Eslava, Salvador; Agnoli, Stefano; Mattevi, Cecilia; Saiz, Eduardo

    2014-07-01

    The widespread technological introduction of graphene beyond electronics rests on our ability to assemble this two-dimensional building block into three-dimensional structures for practical devices. To achieve this goal we need fabrication approaches that are able to provide an accurate control of chemistry and architecture from nano to macroscopic levels. Here, we describe a versatile technique to build ultralight (density ≥1 mg cm-3) cellular networks based on the use of soft templates and the controlled segregation of chemically modified graphene to liquid interfaces. These novel structures can be tuned for excellent conductivity; versatile mechanical response (elastic-brittle to elastomeric, reversible deformation, high energy absorption) and organic absorption capabilities (above 600 g per gram of material). The approach can be used to uncover the basic principles that will guide the design of practical devices that by combining unique mechanical and functional performance will generate new technological opportunities.

  5. Mesoscale assembly of chemically modified graphene into complex cellular networks

    PubMed Central

    Barg, Suelen; Perez, Felipe Macul; Ni, Na; do Vale Pereira, Paula; Maher, Robert C.; Garcia-Tuñon, Esther; Eslava, Salvador; Agnoli, Stefano; Mattevi, Cecilia; Saiz, Eduardo

    2014-01-01

    The widespread technological introduction of graphene beyond electronics rests on our ability to assemble this two-dimensional building block into three-dimensional structures for practical devices. To achieve this goal we need fabrication approaches that are able to provide an accurate control of chemistry and architecture from nano to macroscopic levels. Here, we describe a versatile technique to build ultralight (density ≥1 mg cm−3) cellular networks based on the use of soft templates and the controlled segregation of chemically modified graphene to liquid interfaces. These novel structures can be tuned for excellent conductivity; versatile mechanical response (elastic-brittle to elastomeric, reversible deformation, high energy absorption) and organic absorption capabilities (above 600 g per gram of material). The approach can be used to uncover the basic principles that will guide the design of practical devices that by combining unique mechanical and functional performance will generate new technological opportunities. PMID:24999766

  6. The role of Hsp90 in protein complex assembly.

    PubMed

    Makhnevych, Taras; Houry, Walid A

    2012-03-01

    Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90). PMID:21945180

  7. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  8. (19)F-modified proteins and (19)F-containing ligands as tools in solution NMR studies of protein interactions.

    PubMed

    Sharaf, Naima G; Gronenborn, Angela M

    2015-01-01

    (19)F solution NMR is a powerful and versatile tool to study protein structure and protein-ligand interactions due to the favorable NMR characteristics of the (19)F atom, its absence in naturally occurring biomolecules, and small size. Protocols to introduce (19)F atoms into both proteins and their ligands are readily available and offer the ability to conduct protein-observe (using (19)F-labeled proteins) or ligand-observe (using (19)F-containing ligands) NMR experiments. This chapter provides two protocols for the (19)F-labeling of proteins, using an Escherichia coli expression system: (i) amino acid type-specific incorporation of (19)F-modified amino acids and (ii) site-specific incorporation of (19)F-modified amino acids using recombinantly expressed orthogonal amber tRNA/tRNA synthetase pairs. In addition, we discuss several applications, involving (19)F-modified proteins and (19)F-containing ligands.

  9. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  10. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  11. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  12. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

  13. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

  14. Modified spontaneous emissions of europium complex in weak PMMA opals.

    PubMed

    Wang, Wei; Song, Hongwei; Bai, Xue; Liu, Qiong; Zhu, Yongsheng

    2011-10-28

    Engineering spontaneous emission by means of photonic crystals (PHC) is under extensive study. However PHC modification of line emissions of rare earth (RE) ions has not been thoroughly understood, especially in cases of weak opal PHCs and while emitters are well dispersed into dielectric media. In this study, poly-methyl methacrylate (PMMA) opal PHCs containing uniformly dispersed europium chelate were fabricated with finely controlled photonic stop band (PSB) positions. Measurements of luminescent dynamics and angle resolved/integrated emission spectra as well as numerical calculations of total densities of states (DOS) were performed. We determined that in weak opals, the total spontaneous emission rate (SER) of Σ(5)D(0)-(7)F(J) for Eu(3+) was independent of PSB positions but was higher than that of the disordered powder sample, which was attributed to higher effective refractive indices in the PHC rather than PSB effect. Branch SER of (5)D(0)-(7)F(2) for Eu(3+) in the PHCs, on the other hand, was spatially redistributed, suppressed or enhanced in directions of elevated or reduced optical modes, keeping the angle-integrated total unchanged. All the results are in agreement with total DOS approximation. Our paper addressed two unstudied issues regarding modified narrow line emission in weak opal PHCs: firstly whether PSB could change the SER of emitters and whether there exist, apart from PSB, other reasons to change SERs; secondly, while directional enhancement and suppression by PSB has been confirmed, whether the angle-integrated overall effect is enhancing or suppressing. PMID:21938288

  15. Modified spontaneous emissions of europium complex in weak PMMA opals.

    PubMed

    Wang, Wei; Song, Hongwei; Bai, Xue; Liu, Qiong; Zhu, Yongsheng

    2011-10-28

    Engineering spontaneous emission by means of photonic crystals (PHC) is under extensive study. However PHC modification of line emissions of rare earth (RE) ions has not been thoroughly understood, especially in cases of weak opal PHCs and while emitters are well dispersed into dielectric media. In this study, poly-methyl methacrylate (PMMA) opal PHCs containing uniformly dispersed europium chelate were fabricated with finely controlled photonic stop band (PSB) positions. Measurements of luminescent dynamics and angle resolved/integrated emission spectra as well as numerical calculations of total densities of states (DOS) were performed. We determined that in weak opals, the total spontaneous emission rate (SER) of Σ(5)D(0)-(7)F(J) for Eu(3+) was independent of PSB positions but was higher than that of the disordered powder sample, which was attributed to higher effective refractive indices in the PHC rather than PSB effect. Branch SER of (5)D(0)-(7)F(2) for Eu(3+) in the PHCs, on the other hand, was spatially redistributed, suppressed or enhanced in directions of elevated or reduced optical modes, keeping the angle-integrated total unchanged. All the results are in agreement with total DOS approximation. Our paper addressed two unstudied issues regarding modified narrow line emission in weak opal PHCs: firstly whether PSB could change the SER of emitters and whether there exist, apart from PSB, other reasons to change SERs; secondly, while directional enhancement and suppression by PSB has been confirmed, whether the angle-integrated overall effect is enhancing or suppressing.

  16. Physicochemical properties of protein-modified silver nanoparticles in seawater

    NASA Astrophysics Data System (ADS)

    Zhong, Hangyue

    2013-10-01

    This study investigated the physicochemical properties of silver nanoparticles stabilized with casein protein in seawater. UV?vis spectrometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied to measure the stability of silver nanoparticles in seawater samples. The obtained results show an increased aggregation tendency of silver nanoparticles in seawater, which could be attributed its relatively high cation concentration that could neutralize the negatively charges adsorbed on the surface of silver nanoparticles and reduce the electrostatic repulsion forces between nanoparticles. Similarly, due to the surface charge screening process, the zeta potential of silver nanoparticles in seawater decreased. This observation further supported the aggregation behavior of silver nanoparticles. This study also investigated the dissolution of silver nanoparticles in seawater. Result shows that the silver nanoparticle dissolution in DI water is lower than in seawater, which is attributed to the high Cl? concentration present in seawater. As Cl? can react with silver and form soluble AgCl complex, dissolution of silver nanoparticles was enhanced. Finally, this study demonstrated that silver nanoparticles are destabilized in seawater condition. These results may be helpful in understanding the environmental risk of discharged silver nanoparticles in seawater conditions.

  17. Studying protein complexes by the yeast two-hybrid system.

    PubMed

    Rajagopala, Seesandra V; Sikorski, Patricia; Caufield, J Harry; Tovchigrechko, Andrey; Uetz, Peter

    2012-12-01

    Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

  18. Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

    PubMed

    Mathesius, C A; Barnett, J F; Cressman, R F; Ding, J; Carpenter, C; Ladics, G S; Schmidt, J; Layton, R J; Zhang, J X Q; Appenzeller, L M; Carlson, G; Ballou, S; Delaney, B

    2009-12-01

    Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30s) and intestinal fluid (<1min). The enzymatic activity was completely inactivated at 50 degrees C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.

  19. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  20. Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination.

    PubMed

    Kantake, Noriko; Madiraju, Murty V V M; Sugiyama, Tomohiko; Kowalczykowski, Stephen C

    2002-11-26

    We present biochemical evidence for the functional similarity of Escherichia coli RecO protein and bacteriophage T4 UvsY protein to eukaryotic Rad52 protein. Although Rad52 protein is conserved in eukaryotes, no sequence homologue has been found in prokaryotes or archeabacteria. Rad52 protein has two unique activities: facilitation of replication protein-A (RPA) displacement by Rad51 protein and annealing of RPA-single-stranded DNA (ssDNA) complexes. Both activities require species-specific interaction between Rad52 protein and RPA. Both RecO and UvsY proteins also possess the former property with regard to their cognate ssDNA-binding protein. Here, we report that RecO protein anneals ssDNA that is complexed with only its cognate ssDNA-binding protein, suggesting the involvement of species-specific interactions. Optimal activity for RecO protein occurs after formation of a 1:1 complex with SSB protein. RecR protein, which is known to stimulate RecO protein to facilitate SSB protein displacement by RecA protein, inhibits annealing by RecO protein, suggesting that RecR protein may regulate the choice between the DNA strand invasion versus annealing pathways. In addition, we show that UvsY protein anneals ssDNA; furthermore, ssDNA, which is complexed only with its cognate ssDNA-binding protein, is annealed in the presence of UvsY protein. These results indicate that RecO and possibly UvsY proteins are functional counterparts of Rad52 protein. Based on the conservation of these functions, we propose a modified double-strand break repair model that includes DNA annealing as an important intermediate step. PMID:12438681

  1. Accumulation of small protein molecules in a macroscopic complex coacervate.

    PubMed

    Lindhoud, Saskia; Claessens, Mireille M A E

    2016-01-14

    To obtain insight into the accumulation of proteins into macroscopic complex coacervate phases, the lysozyme concentration in complex coacervates containing the cationic polyelectrolyte poly-(N,N dimethylaminoethyl methacrylate) and the anionic polyelectrolyte polyacrylic acid was investigated as a function of the mixing ratio, protein concentration and ionic strength. Maximal protein enrichment of the complex coacervate phase was observed to require the presence of all three macromolecules. Under optimized conditions the protein concentrations in the complex coacervate were as high as 200 g L(-1). Such high concentrations are comparable to the protein concentration in the cytosol, suggesting that these interesting liquid phases may serve a suitable model system for the phase behavior of the cytosol and genesis and function of membrane-less organelles. The high stability of the complexes and the salt dependent uptake of protein suggest that complex coacervates may provide a way to store hydrated proteins at high concentrations and might therefore be of interest in the formulation of high protein foods.

  2. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  3. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  4. Detection and identification of protein citrullination in complex biological systems.

    PubMed

    Clancy, Kathleen W; Weerapana, Eranthie; Thompson, Paul R

    2016-02-01

    Protein citrullination is a post-translational modification of arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated proteins as well as the specific residues modified within those proteins.

  5. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

    PubMed Central

    Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

    2015-01-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

  6. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

    NASA Astrophysics Data System (ADS)

    Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

    2016-05-01

    Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

  7. Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

    PubMed Central

    Fritz, Kristofer S.; Green, Michelle F.; Petersen, Dennis R.; Hirschey, Matthew D.

    2013-01-01

    Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated metabolism, induce specific

  8. Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

    PubMed

    Brekhman, Vera; Lugassie, Jennie; Zaffryar-Eilot, Shelly; Sabo, Edmond; Kessler, Ofra; Smith, Victoria; Golding, Hana; Neufeld, Gera

    2011-01-01

    Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

  9. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.

  10. Rho-modifying bacterial protein toxins from Photorhabdus species.

    PubMed

    Jank, Thomas; Lang, Alexander E; Aktories, Klaus

    2016-06-15

    Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by N-acetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins.

  11. Protein Connectivity in Chemotaxis Receptor Complexes

    PubMed Central

    Eismann, Stephan; Endres, Robert G.

    2015-01-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  12. Structural study of coacervation in protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    Chodankar, S.; Aswal, V. K.; Kohlbrecher, J.; Vavrin, R.; Wagh, A. G.

    2008-09-01

    Coacervation is a dense liquid-liquid phase separation and herein we report coacervation of protein bovine serum albumin (BSA) in the presence of polyelectrolyte sodium polystyrene sulfonate (NaPSS) under varying solution conditions. Small-angle neutron scattering (SANS) measurements have been performed on above protein-polyelectrolyte complexes to study the structural evolution of the process that leads to coacervation and the phase separated coacervate as a function of solution pH , protein-polyelectrolyte ratio and ionic strength. SANS study prior to phase separation on the BSA-NaPSS complex shows a fractal structure representing a necklace model of protein macromolecules randomly distributed along the polystyrene sulfonate chain. The fractal dimension of the complex decreases as pH is shifted away from the isoelectric point (˜4.7) of BSA protein, which indicates the decrease in the compactness of the complex structure due to increase in the charge repulsion between the protein macromolecules bound to the polyelectrolyte. Concentration-dependence studies of the polyelectrolyte in the complex suggest coexistence of two populations of polyelectrolytes, first one fully saturated with proteins and another one free from proteins. Coacervation phase has been obtained through the turbidity measurement by varying pH of the aqueous solution containing protein and polyelectrolyte from neutral to acidic regime to get them to where the two components are oppositely charged. The spontaneous formation of coacervates is observed for pH values less than 4. SANS study on coacervates shows two length scales related to complex aggregations (mesh size and overall extent of the complex) hierarchically branched to form a larger network. The mesh size represents the distance between cross-linked points in the primary complex, which decreases with increase in ionic strength and remains the same on varying the protein-polyelectrolyte ratio. On the other hand, the overall extent of the

  13. Characteristics and safety assessment of intractable proteins in genetically modified crops.

    PubMed

    Bushey, Dean F; Bannon, Gary A; Delaney, Bryan F; Graser, Gerson; Hefford, Mary; Jiang, Xiaoxu; Lee, Thomas C; Madduri, Krishna M; Pariza, Michael; Privalle, Laura S; Ranjan, Rakesh; Saab-Rincon, Gloria; Schafer, Barry W; Thelen, Jay J; Zhang, John X Q; Harper, Marc S

    2014-07-01

    Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

  14. Blotting protein complexes from native gels to electron microscopy grids.

    PubMed

    Knispel, Roland Wilhelm; Kofler, Christine; Boicu, Marius; Baumeister, Wolfgang; Nickell, Stephan

    2012-01-08

    We report a simple and generic method for the direct transfer of protein complexes separated by native gel electrophoresis to electron microscopy grids. After transfer, sufficient material remains in the gel for identification and characterization by mass spectrometry. The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum.

  15. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  16. Coupling protein complex analysis to peptide based proteomics.

    PubMed

    Gao, Qiang; Madian, Ashraf G; Liu, Xiuping; Adamec, Jiri; Regnier, Fred E

    2010-12-01

    Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

  17. Treatment of hides with tara-modified protein products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In prior research, we demonstrated that gelatin could be modified with quebracho to produce products whose physicochemical properties would enable them to be used effectively as fillers in leather processing, and that leather resulting from this treatment had improved subjective properties with litt...

  18. Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

    PubMed

    Sui, Dexin; Xu, Xinjing; Ye, Xuemei; Liu, Mengyu; Mianecki, Maxwell; Rattanasinchai, Chotirat; Buehl, Christopher; Deng, Xiexiong; Kuo, Min-Hao

    2015-01-01

    Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

  19. Method For Determining And Modifying Protein/Peptide Solubilty

    DOEpatents

    Waldo, Geoffrey S.

    2005-03-15

    A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

  20. Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins.

    PubMed

    Yamauchi, Yasuo; Sugimoto, Yukihiro

    2010-04-01

    Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSIIOEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSIIOEE were modified, but when only OEC33 or PSIIOEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40 degrees C but not at 25 degrees C. In spinach leaves treated at 40 degrees C under light, maximal efficiency of PSII photochemistry (F(v)/F(m) ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40 degrees C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.

  1. Visualizing active membrane protein complexes by electron cryotomography

    PubMed Central

    Gold, Vicki A.M.; Ieva, Raffaele; Walter, Andreas; Pfanner, Nikolaus; van der Laan, Martin; Kühlbrandt, Werner

    2014-01-01

    Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. PMID:24942077

  2. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  3. Sonochemical synthesis of (3-aminopropyl)triethoxysilane-modified monodispersed silica nanoparticles for protein immobilization

    SciTech Connect

    Shen, Shou-Cang; Ng, Wai Kiong; Chia, Leonard; Dong, Yuan-Cai; Tan, Reginald B.H.

    2011-10-15

    Graphical abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by rapid sonochemical co-condensation to achieve high capability for protein immobilization. Highlights: {yields} Amino-modified monodispersed silica nanoparticles were synthesized by rapid co-condensation. {yields} Strong positive charge was created by aminopropyl-modification. {yields} Capability for immobilization of negatively charged protein was enhanced. {yields} Electrostatic interaction between proteins and surface contributed to the enhanced adsorption. -- Abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by a rapid sonochemical co-condensation synthesis procedure. The chemical nature of surface organic modifier on the obtained modified silica nanoparticle was characterized by {sup 13}C and {sup 29}Si MAS Nuclear Magnetic Resonance (NMR) spectroscopies, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)- differential scanning calorimetry (DSC). Due to the strengthened positive surface charge of the silica nanoparticles by the modification with aminopropyl groups, the capability for bovine serum albumin (BSA) adsorption was significantly increased as compared with bare silica nanoparticles. 80 mg/g BSA was adsorbed on modified silica nanoparticles, whereas only 20 mg/g BSA could be loaded on pure silica nanoparticles. The enhanced positive surface charge repelled proteins with net positive charge and the modified silica nanoparticles exhibited negligible adsorption of lysozyme, thus a selective adsorption of proteins could be achieved.

  4. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  5. A Diatom Light-Harvesting Pigment-Protein Complex 1

    PubMed Central

    Friedman, Alan L.; Alberte, Randall S.

    1984-01-01

    A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species. Images Fig. 2 Fig. 3 PMID:16663869

  6. Rho-modifying bacterial protein toxins from Photorhabdus species.

    PubMed

    Jank, Thomas; Lang, Alexander E; Aktories, Klaus

    2016-06-15

    Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by N-acetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins. PMID:26026623

  7. Complexation between dodecyl sulfate surfactant and zein protein in solution.

    PubMed

    Ruso, Juan M; Deo, Namita; Somasundaran, P

    2004-10-12

    Interactions between sodium dodecyl sulfate and zein protein, a model system for the understanding of the effect of surfactants on skin, were investigated using a range of techniques involving UV-vis spectroscopy, TOC (total organic carbon analysis), electrophoresis, and static and dynamic light scattering. Zein protein was solubilized by SDS. The adsorption of SDS onto insoluble protein fraction caused the zeta potential of the complex to become more negative. From these values, we calculated the Gibbs energy of absorption, which decreases when the SDS concentration is raised. Finally the structure of the complex, based on the analysis by static and dynamic light scattering, is proposed to be rod like.

  8. Protein Complex Production from the Drug Discovery Standpoint.

    PubMed

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  9. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  10. Transcriptional regulation of protein complexes within and across species.

    PubMed

    Tan, Kai; Shlomi, Tomer; Feizi, Hoda; Ideker, Trey; Sharan, Roded

    2007-01-23

    Yeast two-hybrid and coimmunoprecipitation experiments have defined large-scale protein-protein interaction networks for many model species. Separately, systematic chromatin immunoprecipitation experiments have enabled the assembly of large networks of transcriptional regulatory interactions. To investigate the functional interplay between these two interaction types, we combined both within a probabilistic framework that models the cell as a network of transcription factors regulating protein complexes. This framework identified 72 putative coregulated complexes in yeast and allowed the prediction of 120 previously uncharacterized transcriptional interactions. Several predictions were tested by new microarray profiles, yielding a confirmation rate (58%) comparable with that of direct immunoprecipitation experiments. Furthermore, we extended our framework to a cross-species setting, identifying 24 coregulated complexes that were conserved between yeast and fly. Analyses of these conserved complexes revealed different conservation levels of their regulators and provided suggestive evidence that protein-protein interaction networks may evolve more slowly than transcriptional interaction networks. Our results demonstrate how multiple molecular interaction types can be integrated toward a global wiring diagram of the cell, and they provide insights into the evolutionary dynamics of protein complex regulation.

  11. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  12. Proteins immobilization on the surface of modified plant viral particles coated with hydrophobic polycations.

    PubMed

    Nikitin, Nikolai A; Malinin, Andrei S; Trifonova, Ekaterina A; Rakhnyanskaya, Anna A; Yaroslavov, Aleksandr A; Karpova, Olga V; Atabekov, Joseph G

    2014-01-01

    Two hydrophobic cations based on poly-N-ethyl-vinylpyridine were used to produce biologically active complexes. The complexes obtained from tobacco mosaic virus (TMV) spherical particles (SPs), hydrophobic polycation, and a model protein were stable and did not aggregate in solution, particularly at high ionic strengths. The nucleic acid-free SPs were generated by thermal remodeling of the TMV (helical rod-shaped plant virus). The model protein preserved its antigenic activity in the ternary complex (SP-polycation-protein). Immobilization of proteins on the surface of SPs coated with hydrophobic cation is a promising approach to designing biologically active complexes used in bionanotechnologies. PMID:25121344

  13. Reversible and oriented immobilization of ferrocene-modified proteins.

    PubMed

    Yang, Lanti; Gomez-Casado, Alberto; Young, Jacqui F; Nguyen, Hoang D; Cabanas-Danés, Jordi; Huskens, Jurriaan; Brunsveld, Luc; Jonkheijm, Pascal

    2012-11-21

    Adopting supramolecular chemistry for immobilization of proteins is an attractive strategy that entails reversibility and responsiveness to stimuli. The reversible and oriented immobilization and micropatterning of ferrocene-tagged yellow fluorescent proteins (Fc-YFPs) onto β-cyclodextrin (βCD) molecular printboards was characterized using surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in combination with electrochemistry. The proteins were assembled on the surface through the specific supramolecular host-guest interaction between βCD and ferrocene. Application of a dynamic covalent disulfide lock between two YFP proteins resulted in a switch from monovalent to divalent ferrocene interactions with the βCD surface, yielding a more stable protein immobilization. The SPR titration data for the protein immobilization were fitted to a 1:1 Langmuir-type model, yielding K(LM) = 2.5 × 10(5) M(-1) and K(i,s) = 1.2 × 10(3) M(-1), which compares favorably to the intrinsic binding constant presented in the literature for the monovalent interaction of ferrocene with βCD self-assembled monolayers. In addition, the SPR binding experiments were qualitatively simulated, confirming the binding of Fc-YFP in both divalent and monovalent fashion to the βCD monolayers. The Fc-YFPs could be patterned on βCD surfaces in uniform monolayers, as revealed using fluorescence microscopy and atomic force microscopy measurements. Both fluorescence microscopy imaging and SPR measurements were carried out with the in situ capability to perform cyclic voltammetry and chronoamperometry. These studies emphasize the repetitive desorption and adsorption of the ferrocene-tagged proteins from the βCD surface upon electrochemical oxidation and reduction, respectively.

  14. Monte Carlo Modeling of Spectral Diffusion Employing Multiwell Protein Energy Landscapes: Application to Pigment-Protein Complexes Involved in Photosynthesis.

    PubMed

    Najafi, Mehdi; Zazubovich, Valter

    2015-06-25

    We are reporting development and initial applications of the light-induced and thermally induced spectral diffusion modeling software, covering nonphotochemical spectral hole burning (NPHB), hole recovery, and single-molecule spectroscopy and involving random generation of the multiwell protein energy landscapes. The model includes tunneling and activated barrier-hopping in both ground and excited states of a protein-chromophore system. Evolution of such a system is predicted by solving the system of rate equations. Using the barrier parameters from the range typical for the energy landscapes of the pigment-protein complexes involved in photosynthesis, we (a) show that realistic cooling of the sample must result in proteins quite far from thermodynamic equilibrium, (b) demonstrate hole evolution in the cases of burning, fixed-temperature recovery and thermocycling that mostly agrees with the experiment and modeling based on the NPHB master equation, and (c) explore the effects of different protein energy landscapes on the antihole shape. Introducing the multiwell energy landscapes and starting the hole burning experiments in realistic nonequilibrium conditions are not sufficient to explain all experimental observations even qualitatively. Therefore, for instance, one is required to invoke the modified NPHB mechanism where a complex interplay of several small conformational changes is poising the energy landscape of the pigment-protein system for downhill tunneling.

  15. A Local Discontinuous Galerkin Method for the Complex Modified KdV Equation

    SciTech Connect

    Li Wenting; Jiang Kun

    2010-09-30

    In this paper, we develop a local discontinuous Galerkin(LDG) method for solving complex modified KdV(CMKdV) equation. The LDG method has the flexibility for arbitrary h and p adaptivity. We prove the L{sup 2} stability for general solutions.

  16. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  17. Protein- and Peptide-Modified Synthetic Polymeric Biomaterials

    PubMed Central

    Krishna, Ohm D.; Kiick, Kristi L.

    2015-01-01

    This review presents an overview on biohybrid approaches of integrating the structural and functional features of proteins and peptides with synthetic polymers and the resulting unique properties in such hybrids, with a focus on bioresponsive/bioactive systems with biomaterials applications. The review is divided in two broad sections. First, we describe several examples of biohybrids produced by combining versatile synthetic polymers with proteins/enzymes and drugs that have resulted in (1) hybrid materials based on responsive polymers, (2) responsive hydrogels based on enzyme-catalyzed reactions, protein–protein interactions and protein–drug sensing, and (3) dynamic hydrogels based on conformational changes of a protein. Next, we present hybrids produced by combining synthetic polymers with peptides, classified based on the properties of the peptide domain: (1) peptides with different conformations, such as α-helical, coiled-coil, and β-sheet; (2) peptides derived from structural protein domains such as silk, elastin, titin, and collagen; and (3) peptides with other biofunctional properties such as cell-binding domains and enzyme-recognized degradation domains. PMID:20091878

  18. The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants1[OPEN

    PubMed Central

    Carr, Craig; Asensi-Fabado, Maria A.; Donald, Naomi A.; Hannah, Matthew A.; Amtmann, Anna

    2016-01-01

    Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes. PMID:26951436

  19. The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants.

    PubMed

    Perrella, Giorgio; Carr, Craig; Asensi-Fabado, Maria A; Donald, Naomi A; Páldi, Katalin; Hannah, Matthew A; Amtmann, Anna

    2016-05-01

    Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

  20. Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo

    PubMed Central

    Katsumi, Hidemasa; Sano, Jun-ichi; Nishikawa, Makiya; Hanzawa, Keiko; Sakane, Toshiyasu; Yamamoto, Akira

    2015-01-01

    To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight—yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)—were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (111In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of 111In-ALN-modified proteins were proportional to the degree of ALN modification. 111In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for 111In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. 111In-SOD modified with both polyethylene glycol (PEG) and ALN (111In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance. PMID:26287482

  1. Rnq1: an epigenetic modifier of protein function in yeast.

    PubMed

    Sondheimer, N; Lindquist, S

    2000-01-01

    Two protein-based genetic elements (prions) have been identified in yeast. It is not clear whether other prions exist, nor is it understood how one might find them. We established criteria for searching protein databases for prion candidates and found several. The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble. The insoluble state is dominant and transmitted between cells through the cytoplasm. When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion [PSI+], the phenotypic and epigenetic behavior of [PSI+] was fully recapitulated. These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions.

  2. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

    Barr, E.; Leiden, J.M. )

    1991-12-06

    The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.

  3. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  4. Chromatographic and traditional albumin isotherms on cellulose: a model for wound protein adsorption on modified cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Albumin is the most abundant protein found in healing wounds. Traditional and chromatogrpahic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressin...

  5. Modification of recombinant maize ChitA chitinase by fungal chitinase-modifying proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In commercial maize, there are at least two different alleles of the chiA gene that encode alloforms of ChitA chitinase, a protein that is abundant in developing seed. Both known alloforms are modified by Bz-cmp, a protein secreted by the fungal pathogen Bipolaris zeicola. One alloform (ChitA-B73) i...

  6. Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA.

    PubMed

    Hinerman, Jennifer M; Dignam, J David; Mueser, Timothy C

    2012-05-25

    Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).

  7. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    SciTech Connect

    Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).

  8. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  9. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce.

    PubMed

    Sun, Hyeon-Jin; Cui, Min-Long; Ma, Biao; Ezura, Hiroshi

    2006-01-23

    Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein. PMID:16406368

  10. Biocontainment of genetically modified organisms by synthetic protein design.

    PubMed

    Mandell, Daniel J; Lajoie, Marc J; Mee, Michael T; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E; Gregg, Christopher J; Stoddard, Barry L; Church, George M

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites. PMID:25607366

  11. Biocontainment of genetically modified organisms by synthetic protein design.

    PubMed

    Mandell, Daniel J; Lajoie, Marc J; Mee, Michael T; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E; Gregg, Christopher J; Stoddard, Barry L; Church, George M

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

  12. Biocontainment of genetically modified organisms by synthetic protein design

    NASA Astrophysics Data System (ADS)

    Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T.; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E.; Gregg, Christopher J.; Stoddard, Barry L.; Church, George M.

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

  13. Biocontainment of genetically modified organisms by synthetic protein design

    PubMed Central

    Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T.; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E.; Gregg, Christopher J.; Stoddard, Barry L.; Church, George M.

    2015-01-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient either because they impose evolutionary pressure on the organism to eject the safeguard, because they can be circumvented by environmentally available compounds, or because they can be overcome by horizontal gene transfer (HGT). Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code to confer metabolic dependence on nonstandard amino acids for survival. The resulting GMOs cannot metabolically circumvent their biocontainment mechanisms using environmentally available compounds, and they exhibit unprecedented resistance to evolutionary escape via mutagenesis and HGT. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by reliance on synthetic metabolites. PMID:25607366

  14. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  15. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  16. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    PubMed

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  17. Synthetic RNA-protein complex shaped like an equilateral triangle

    NASA Astrophysics Data System (ADS)

    Ohno, Hirohisa; Kobayashi, Tetsuhiro; Kabata, Rinko; Endo, Kei; Iwasa, Takuma; Yoshimura, Shige H.; Takeyasu, Kunio; Inoue, Tan; Saito, Hirohide

    2011-02-01

    Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ~60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

  18. Solid-State NMR Spectroscopy of Protein Complexes

    PubMed Central

    Sun, Shangjin; Han, Yun; Paramasivam, Sivakumar; Yan, Si; Siglin, Amanda E.; Williams, John C.; Byeon, In-Ja L.; Ahn, Jinwoo; Gronenborn, Angela M.; Polenova, Tatyana

    2016-01-01

    Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR based methods to study several protein complexes. In this chapter, we discuss the general solid-state NMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection and data analysis. PMID:22167681

  19. Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction.

    PubMed

    Gendron, Joshua M; Webb, Kristofor; Yang, Bing; Rising, Lisa; Zuzow, Nathan; Bennett, Eric J

    2016-08-01

    Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations.

  20. Structural and evolutionary versatility in protein complexes with uneven stoichiometry.

    PubMed

    Marsh, Joseph A; Rees, Holly A; Ahnert, Sebastian E; Teichmann, Sarah A

    2015-03-16

    Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric complexes of known structure have even stoichiometry, a significant minority have uneven stoichiometry--that is, differing numbers of each subunit type. To adopt this uneven stoichiometry, sequence-identical subunits must be asymmetric with respect to each other, forming different interactions within the complex. Here we first investigate the occurrence of uneven stoichiometry, demonstrating that it is common in vitro and is likely to be common in vivo. Next, we elucidate the structural determinants of uneven stoichiometry, identifying six different mechanisms by which it can be achieved. Finally, we study the frequency of uneven stoichiometry across evolution, observing a significant enrichment in bacteria compared with eukaryotes. We show that this arises due to a general increased tendency for bacterial proteins to self-assemble and form homomeric interactions, even within the context of a heteromeric complex.

  1. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  2. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  3. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT. PMID:22118932

  4. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  5. Discovery of host-viral protein complexes during infection

    PubMed Central

    Rowles, Daniell L.; Terhune, Scott S.; Cristea, Ileana M.

    2014-01-01

    Summary Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally-regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus-host protein interactions and protein complexes, which we have successfully utilized to characterize spatial-temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems. PMID:23996249

  6. Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes.

    PubMed

    Ataie, G; Moosavi-Movahedi, A A; Saboury, A A; Hakimelahi, G H; Hwu, J R; Tsay, S C

    2000-03-16

    Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18000, 22000 and 21900 kJ mol(-1) respectively.

  7. Functional expression of miraculin, a taste-modifying protein in Escherichia coli.

    PubMed

    Matsuyama, Tomomi; Satoh, Makiko; Nakata, Rieko; Aoyama, Takashi; Inoue, Hiroyasu

    2009-04-01

    Miraculin isolated from red berries of Richadella dulcifica, a native shrub of West Africa, has the unusual property of modifying a sour taste into a sweet one. This homodimer protein consists of two glycosylated polypeptides that are cross-linked by a disulfide bond. Recently, functional expression of miraculin was reported in host cells with the ability to glycosylate proteins, such as lettuce, tomato and the microbe Aspergillus oryzae, but not Escherichia coli. Thus, a question remains as to whether glycosylation of miraculin is essential for its taste-modifying properties. Here we show that recombinant miraculin expressed in E. coli has taste-modifying properties as a homodimer, not as a monomer, indicating that glycosylation is not essential for the taste-modifying property. PMID:19122203

  8. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    PubMed

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  9. Relationship between oxygen concentration, shear force and protein oxidation in modified atmosphere packaged pork.

    PubMed

    Bao, Yulong; Ertbjerg, Per

    2015-12-01

    Pork loins were stored at 5°C for 14 days to investigate the effect of oxygen concentration in modified atmosphere packaging (MAP) on shear force and oxidation of lipids and proteins. The modified atmosphere contained 0 to 80% O2, 20% CO2, and balanced with N2. The results showed that shear force and thiobarbituric acid-reactive substances (TBARS) values increased with increasing oxygen concentration. Protein oxidation when measured as loss of free thiol groups, was greater in meat packaged under oxygen (20-80%). Myosin heavy chain (MHC) cross-linking, another marker of protein oxidation, was greater in MAP with 80% oxygen than 0% and 20% oxygen. Desmin degradation was not affected by the presence of oxygen, suggesting that the mechanism of oxygen-induced toughening of meat is through protein oxidation leading to cross-linking of structural proteins rather than through inactivation of proteolytic enzymes leading to reduced proteolysis.

  10. Maltodextrin-modified magnetic microspheres for selective enrichment of maltose binding proteins.

    PubMed

    Zheng, Jin; Ma, Chongjun; Sun, Yangfei; Pan, Miaorong; Li, Li; Hu, Xiaojian; Yang, Wuli

    2014-03-12

    In this work, maltodextrin-modified magnetic microspheres Fe3O4@SiO2-Maltodextrin (Fe3O4@SiO2-MD) with uniform size and fine morphology were synthesized through a facile and low-cost method. As the maltodextrins on the surface of microspheres were combined with maltose binding proteins (MBP), the magnetic microspheres could be applied to enriching standard MBP fused proteins. Then, the application of Fe3O4@SiO2-MD in one-step purification and immobilization of MBP fused proteins was demonstrated. For the model protein we examined, Fe3O4@SiO2-MD showed excellent binding selectivity and capacity against other Escherichia coli proteins in the crude cell lysate. Additionally, the maltodextrin-modified magnetic microspheres can be recycled for several times without significant loss of binding capacity.

  11. Relationship between oxygen concentration, shear force and protein oxidation in modified atmosphere packaged pork.

    PubMed

    Bao, Yulong; Ertbjerg, Per

    2015-12-01

    Pork loins were stored at 5°C for 14 days to investigate the effect of oxygen concentration in modified atmosphere packaging (MAP) on shear force and oxidation of lipids and proteins. The modified atmosphere contained 0 to 80% O2, 20% CO2, and balanced with N2. The results showed that shear force and thiobarbituric acid-reactive substances (TBARS) values increased with increasing oxygen concentration. Protein oxidation when measured as loss of free thiol groups, was greater in meat packaged under oxygen (20-80%). Myosin heavy chain (MHC) cross-linking, another marker of protein oxidation, was greater in MAP with 80% oxygen than 0% and 20% oxygen. Desmin degradation was not affected by the presence of oxygen, suggesting that the mechanism of oxygen-induced toughening of meat is through protein oxidation leading to cross-linking of structural proteins rather than through inactivation of proteolytic enzymes leading to reduced proteolysis. PMID:26241463

  12. Photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex.

    PubMed

    Zhang, Zhigang; Dai, Ruihui; Ma, Jiajia; Wang, Shuying; Wei, Xuehong; Wang, Hongfei

    2015-02-01

    Many planar photosensitizers tend to self-aggregate via van der Waals interactions between π-conjugated systems. The self-aggregation of the photosensitizer may reduce the efficiency of the photosensitizer to generate singlet oxygen, thereby diminishing its photodynamic activity. Efforts have been made to improve the photodynamic activity of bis-(o-diiminobenzosemiquinonato)platinum(II) which has planar geometry by the introduction of the sterically hindered triphenylamine moiety into the ligand. Herein we report the photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex in red light studied by fluorescence spectra, agarose gel assay and cell viability assay. The results suggest that the triphenylamine-modified platinum-diimine complex has better capability to generate singlet oxygen than bis-(o-diiminobenzosemiquinonato)platinum(II), and it can induce DNA damage in red light, causing high photocytotoxicity in HepG-2 cells in vitro.

  13. Evaluation of the Use of Complex Mineral Concentrate as a Modifier Steel

    NASA Astrophysics Data System (ADS)

    Gizatulin, R. A.; Fedoseev, S. N.; Dariev, R. S.

    2016-04-01

    Increasing customer demands for quality of the resulting metal, and in the first place, the impurities, metallurgists dictate need to develop new and improved technologies. Thus, a significant reduction in metal losses can be achieved by developing new complex alloy steels, special purpose, improving technology of their production and developing new technology of smelting to improve the physical, mechanical, foundry and operational characteristics by influencing the structure of the steel by modifying the liquid melt, change more favorable morphology of nonmetallic inclusions. For complex-alloyed steels expensive and scarce alloying elements Ti, Nb, Zr, etc., are used, which are inaccessible to conventional structural steels. In this regard, the paper also presents the results of applying of innovative modifiers containing alloying elements (Ti, Nb, Zr, etc.) based on mineral concentrates in the Tomsk region.

  14. Protein corona - from molecular adsorption to physiological complexity.

    PubMed

    Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

    2015-01-01

    In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.

  15. Modified Hilbert transform pair and Kramers-Kronig relations for complex permittivities

    NASA Technical Reports Server (NTRS)

    Cockrell, C. R.

    1990-01-01

    Modified versions of the Hilbert transform pair and the Kramers-Kronig relations are derived for the complex permittivity of a plasma/dielectric medium which is singular at the frequency of the applied electric field equal to 0. Such a complex permittivity exists when the plasma/dielectric model allows a loss term but no restoring term. Permittivity, in which both loss and restoring terms are included, is shown to satisfy the standard Hilbert transform pair and, thus, the Kramers-Kronig relations.

  16. Study of protein complexes via homology modeling, applied to cysteine proteases and their protein inhibitors.

    PubMed

    Tastan Bishop, Ozlem; Kroon, Matthys

    2011-12-01

    This paper develops and evaluates large-scale calculation of 3D structures of protein complexes by homology modeling as a promising new approach for protein docking. The complexes investigated were papain-like cysteine proteases and their protein inhibitors, which play numerous roles in human and parasitic metabolisms. The structural modeling was performed in two parts. For the first part (evaluation set), nine crystal structure complexes were selected, 1325 homology models of known complexes were rebuilt by various templates including hybrids, allowing an analysis of the factors influencing the accuracy of the models. The important considerations for modeling the interface were protease coverage and inhibitor sequence identity. In the second part (study set), the findings of the evaluation set were used to select appropriate templates to model novel cysteine protease-inhibitor complexes from human and malaria parasites Plasmodium falciparum and Plasmodium vivax. The energy scores, considering the evaluation set, indicate that the models are of high accuracy. PMID:21365221

  17. Identification of a chitinase modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are proteases, secreted by fungal pathogens, which truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. Cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. H...

  18. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  19. How to Build a Complex, Functional Propeller Protein, From Parts.

    PubMed

    Clark, Patricia L

    2016-04-01

    By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed β-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo.

  20. Synthesis of modified cyclic and acyclic dextrins and comparison of their complexation ability

    PubMed Central

    Jicsinszky, László; Sohajda, Tamás; Puskás, István; Fenyvesi, Éva

    2014-01-01

    Summary We compared the complex forming ability of α-, β- and γ-cyclodextrins (α-CD, β-CD and γ-CD) with their open ring analogs. In addition to the native cyclodextrins also modified cyclodextrins and the corresponding maltooligomers, functionalized with neutral 2-hydroxypropyl moieties, were synthesized. A new synthetic route was worked out via bromination, benzylation, deacetylation and debenzylation to obtain the 2-hydroxypropyl maltooligomer counterparts. The complexation properties of non-modified and modified cyclic and acyclic dextrins were studied and compared by photon correlation spectroscopy (PCS) and capillary electrophoresis (CE) using model guest compounds. In some cases cyclodextrins and their open-ring analogs (acyclodextrins) show similar complexation abilities, while with other guests considerably different behavior was observed depending on the molecular dimensions and chemical characteristics of the guests. This was explained by the enhanced flexibility of the non-closed rings. Even the signs of enantiorecognition were observed for the chloropheniramine/hydroxypropyl maltohexaose system. Further studies are planned to help the deeper understanding of the interactions. PMID:25550750

  1. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event. PMID:26079086

  2. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event.

  3. Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

    PubMed Central

    Riechers, Sean-Patrick; Muehlenberg, Katja; Möller, Angeli; Reinhardt, Anita; Vinayagam, Arunachalam; Schaefer, Martin H.; Boutros, Michael; Tricoire, Hervé; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2012-01-01

    Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo. PMID:22916034

  4. DOCK/PIERR: web server for structure prediction of protein-protein complexes.

    PubMed

    Viswanath, Shruthi; Ravikant, D V S; Elber, Ron

    2014-01-01

    In protein docking we aim to find the structure of the complex formed when two proteins interact. Protein-protein interactions are crucial for cell function. Here we discuss the usage of DOCK/PIERR. In DOCK/PIERR, a uniformly discrete sampling of orientations of one protein with respect to the other, are scored, followed by clustering, refinement, and reranking of structures. The novelty of this method lies in the scoring functions used. These are obtained by examining hundreds of millions of correctly and incorrectly docked structures, using an algorithm based on mathematical programming, with provable convergence properties.

  5. Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

    PubMed

    Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

    2016-04-19

    The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein

  6. Polymeric human Fc-fusion proteins with modified effector functions

    NASA Astrophysics Data System (ADS)

    Mekhaiel, David N. A.; Czajkowsky, Daniel M.; Andersen, Jan Terje; Shi, Jianguo; El-Faham, Marwa; Doenhoff, Michael; McIntosh, Richard S.; Sandlie, Inger; He, Jianfeng; Hu, Jun; Shao, Zhifeng; Pleass, Richard J.

    2011-10-01

    The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.

  7. Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

    PubMed Central

    Ponchon, Luc; Catala, Marjorie; Seijo, Bili; El Khouri, Marguerite; Dardel, Frédéric; Nonin-Lecomte, Sylvie; Tisné, Carine

    2013-01-01

    RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA–protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA–His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins. PMID:23804766

  8. Computational large-scale mapping of protein-protein interactions using structural complexes.

    PubMed

    Shoemaker, Benjamin; Wuchty, Stefan; Panchenko, Anna R

    2013-01-01

    Although the identification of protein interactions by high-throughput methods progresses at a fast pace, "interactome" datasets still suffer from high rates of false positives and low coverage. To map the interactome of any organism, this unit presents a computational framework to predict protein-protein or gene-gene interactions utilizing experimentally determined evidence of structural complexes, atomic details of binding interfaces and evolutionary conservation.

  9. Cell separation by immunoaffinity partitioning with polyethylene glycol-modified Protein A in aqueous polymer two-phase systems

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Van Alstine, James M.; Snyder, Robert S.; Shafer, Steven G.; Harris, J. Milton

    1988-01-01

    Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work it is shown that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, the paper provides a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.

  10. Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior

    PubMed Central

    Strambio-de-Castillia, Caterina; Blobel, Günter; Rout, Michael P.

    1999-01-01

    While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC. PMID:10085285

  11. Utilization of modified surfactant-associated protein B for delivery of DNA to airway cells in culture.

    PubMed Central

    Baatz, J E; Bruno, M D; Ciraolo, P J; Glasser, S W; Stripp, B R; Smyth, K L; Korfhagen, T R

    1994-01-01

    Pulmonary surfactant lines the airway epithelium and creates a potential barrier to successful transfection of the epithelium in vivo. Based on the functional properties of pulmonary surfactant protein B (SP-B) and the fact that this protein is neither toxic nor immunogenic in the airway, we hypothesized that SP-B could be modified to deliver DNA to airway cells. We have modified native bovine SP-B by the covalent linkage of poly(lysine) (average molecular mass of 3.3 or 10 kDa) to the N terminus of SP-B and formed complexes between a test plasmid and the modified SP-B. Transfection efficiency was determined by transfection of pulmonary adenocarcinoma cells (H441) in culture with the test plasmid pCPA-RSV followed by measurement of activity of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfections were performed with DNA.protein complexes using poly(lysine)10kDa-SP-B ([Lys]10kDa-SP-B) or poly(lysine)3.3kDa-SP-B ([Lys]3.3kDa-SP-B), and results were compared with transfections using unmodified poly(lysine).DNA, unmodified SP-B.DNA, or DNA only. For [Lys]10kDa-SP-B.pCPA-RSV preparations, CAT activity was readily detectable above the background of [Lys]3.3kDa-SP-B or unmodified SP-B. The SP-B-poly(lysine) conjugates were effective over a broad range of protein-to-DNA molar ratios, although they were optimal at approximately 500:1-1000:1. Transfection efficiency varied with the tested cell line but was not specific to airway cells. Addition of replication-defective adenovirus to the [Lys]10kDa-SP-B.pCPA-RSV complex enhanced CAT activity about 30-fold with respect to that produced by the [Lys]10kDa-SP-B.pCPA-RSV complex alone. This increase suggests routing of the adenoviral.[Lys]10kDa-SP-B.pCPA-RSV complex through an endosomal pathway. Effects of covalent modification on the secondary structure of SP-B were examined by Fourier transform infrared spectrometry (FTIR). Results of FTIR indicated that the conformation of [Lys]10kDa-SP-B was

  12. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins.

    PubMed

    Takata, K; Horiuchi, S; Araki, N; Shiga, M; Saitoh, M; Morino, Y

    1988-10-15

    Long term incubation of proteins with glucose, named the Maillard reaction (Maillard, L. C. (1912) C. R. Acad. Sci. (Paris) 154, 66-68), gives rise to advanced glycosylation end product (AGE) with fluorescence, color, as well as cross-linked properties. The receptor-mediated endocytosis of AGE-proteins by macrophages was reported (Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5588-5592). The present study on the binding of AGE-bovine serum albumin (BSA) to rat peritoneal macrophages and sinusoidal liver cells demonstrated the presence of a saturable, high affinity receptor for AGE-BSA with Kd = 2.4 x 10(-7) M (macrophages) and 2.1 x 10(-7) M (sinusoidal cells). The cellular binding of AGE-BSA and its endocytic uptake by these cells were competitively inhibited by BSA preparations modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, ligands known to be specific for a scavenger receptor for aldehyde-modified proteins (Horiuchi, S., Murakami, M., Takata, K., and Morino, Y. (1986). J. Biol. Chem. 261, 4962-4966). These ligands also had a profound in vivo effect on the plasma clearance of 125I-AGE-BSA as well as its hepatic uptake. Thus, endocytic uptake of AGE-proteins by macrophages appeared to be mediated by a scavenger receptor for aldehyde-modified proteins. This provides evidence for the biological importance of the scavenger receptor in eliminating senescent macromolecules from the circulation.

  13. Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.

    PubMed

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-08-20

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase. PMID:20538592

  14. Structural and Functional Properties of Soy Protein Isolates Modified by Soy Soluble Polysaccharides.

    PubMed

    Xu, Yan-Teng; Liu, Ling-Ling

    2016-09-28

    Aiming to achieve the modification to soy protein isolate (SPI) by soy soluble polysaccharides (SSPS), electrically driven complex systems were first established in the environment of pH 3.0, and then reconstituted SPI particles with different SPI-SSPS ratios were obtained under freeze-drying process. Through this treatment, the structures of SPI particles were partly unfolded and adsorbed SSPS mainly via hydrophobic interactions and hydrogen bonding with larger particle sizes. The adherence of SSPS decreased the surface hydrophobicity of reconstituted SPI particles, but exerted not much influence on the emulsifying and foaming activities and increased the corresponding stabilities due to enhancing the unfolded extent of structure and improving the conformation flexibility. Reconstituted SPI-SSPS particles might rearrange and link each other due to the presence of SSPS on the air-water interface to better stabilize these systems. At SPI-SSPS ratio of 10:1, lower temperature was required to form gels with lower gel intensity and porous structure. The findings provide a further comprehension to the relationship between structures and functional properties of SPI modified by SSPS and the feasibility of applying these reconstituted particles to needed areas. PMID:27608266

  15. Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin.

    PubMed

    Takahashi, N; Hitotsuya, H; Hanzawa, H; Arata, Y; Kurihara, Y

    1990-05-15

    The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text) PMID:2335505

  16. Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

    PubMed

    Yen, Eric A; Tsay, Aaron; Waldispuhl, Jerome; Vogel, Jackie

    2014-05-01

    Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden

  17. Modeling of Protein Binary Complexes Using Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Chance, M.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints--positive and/or negative--in the docking step and are also used to decide the type of energy filter--electrostatics or desolvation--in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.

  18. Identification of O-Linked N-Acetylglucosamine (O-GlcNAc)-modified Osteoblast Proteins by Electron Transfer Dissociation Tandem Mass Spectrometry Reveals Proteins Critical for Bone Formation*

    PubMed Central

    Nagel, Alexis K.; Schilling, Michael; Comte-Walters, Susana; Berkaw, Mary N.; Ball, Lauren E.

    2013-01-01

    The nutrient-responsive β-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFβ-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation. PMID:23443134

  19. Identification of oxidatively modified proteins in salt-stressed Arabidopsis: a carbonyl-targeted proteomics approach.

    PubMed

    Mano, Jun'ichi; Nagata, Mitsuaki; Okamura, Shoutarou; Shiraya, Takeshi; Mitsui, Toshiaki

    2014-07-01

    In plants, environmental stresses cause an increase in the intracellular level of reactive oxygen species (ROS), leading to tissue injury. To obtain biochemical insights into this damage process, we investigated the protein carbonyls formed by ROS or by the lipid peroxide-derived α,β-unsaturated aldehydes and ketones (i.e. reactive carbonyl species, or RCS) in the leaves of Arabidopsis thaliana under salt stress. A. thaliana Col-0 plants that we treated with 300 mM NaCl for 72 h under continuous illumination suffered irreversible leaf damage. Several RCS such as 4-hydroxy-(E)-2-nonenal (HNE) were increased within 12 h of this salt treatment. Immunoblotting using distinct antibodies against five different RCS, i.e. HNE, 4-hydroxy-(E)-2-hexenal, acrolein, crotonaldehyde and malondialdehyde, revealed that RCS-modified proteins accumulated in leaves with the progress of the salt stress treatment. The band pattern of Western blotting suggested that these different RCS targeted a common set of proteins. To identify the RCS targets, we collected HNE-modified proteins via an anti-HNE antiserum affinity trap and performed an isobaric tag for relative and absolute quantitation, as a quantitative proteomics approach. Seventeen types of protein, modified by 2-fold more in the stressed plants than in the non-stressed plants, were identified as sensitive RCS targets. With aldehyde-reactive probe-based affinity trapping, we collected the oxidized proteins and identified 22 additional types of protein as sensitive ROS targets. These RCS and ROS target proteins were distributed in the cytosol and apoplast, as well as in the ROS-generating organelles the peroxisome, chloroplast and mitochondrion, suggesting the participation of plasma membrane oxidation in the cellular injury. Possible mechanisms by which these modified targets cause cell death are discussed.

  20. Changes in protein structure at the interface accompanying complex formation.

    PubMed

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H; Chakrabarti, Pinak

    2015-11-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein-Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

  1. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  2. Mechanism of action of regulatory proteins encoded by complex retroviruses.

    PubMed Central

    Cullen, B R

    1992-01-01

    Complex retroviruses are distinguished by their ability to control the expression of their gene products through the action of virally encoded regulatory proteins. These viral gene products modulate both the quantity and the quality of viral gene expression through regulation at both the transcriptional and posttranscriptional levels. The most intensely studied retroviral regulatory proteins, termed Tat and Rev, are encoded by the prototypic complex retrovirus human immunodeficiency virus type 1. However, considerable information also exists on regulatory proteins encoded by human T-cell leukemia virus type I, as well as several other human and animal complex retroviruses. In general, these data demonstrate that retrovirally encoded transcriptional trans-activators can exert a similar effect by several very different mechanisms. In contrast, posttranscriptional regulation of retroviral gene expression appears to occur via a single pathway that is probably dependent on the recruitment of a highly conserved cellular cofactor. These two shared regulatory pathways are proposed to be critical to the ability of complex retroviruses to establish chronic infections in the face of an ongoing host immune response. Images PMID:1406488

  3. Simultaneous complexation of organic compounds and heavy metals by a modified cyclodextrin

    SciTech Connect

    Wang, X.; Brusseau, M.L.

    1995-10-01

    The cleanup of contaminated soil and groundwater at hazardous waste sites has become a major focus of research and policy debate. A major factor complicating the cleanup of many sites is the cooccurrence of organic compounds and heavy metals, the so-called mixed wastes. We investigated the ability of a modified cyclodextrin to simultaneously complex low-polarity organic compounds and heavy metals. The results of the experiments showed that carboxymethyl-{beta}-cyclodextrin could simultaneously increase the apparent aqueous solubilities of the selected organic compounds (anthracene, trichlorobenzene; biphenyl, and ODT) and complex with Cd{sup 2+}. This complexation was not significantly affected by changes in pH or by the presence of relatively high concentrations of Ca{sup 2+}. It is possible that this reagent can be used successfully to remediate hazardous waste sites contaminated by mixed wastes. 11 refs., 7 figs.

  4. A modified complex modal testing technique for a rotating tire with a flexible ring model

    NASA Astrophysics Data System (ADS)

    Lee, Jongsuh; Wang, Semyung; Pluymers, Bert; Desmet, Wim; Kindt, Peter

    2015-08-01

    Natural frequencies, mode shapes and modal damping values are the most important parameters to describe the noise and vibration behavior of a mechanical system. For rotating machinery, however, the directivity of the propagation wave of each mode should also be taken into account. For rotating systems, this directivity can be determined by complex modal testing. In this paper, a rolling tire is represented as a flexible ring model. The limitation of application of the complex modal testing which requires two directional measurements at a certain point, which is difficult to measure in practice, has been overcome through a modified complex modal testing which requires only one directional measurements at any two points. The technique is described in detail and applied to both a numerical example and to an experimental data set of a real rotating tire.

  5. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  6. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  7. The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.

    PubMed

    Rojas-Benítez, Diego; Ibar, Consuelo; Glavic, Álvaro

    2013-01-01

    The TOR signaling pathway is crucial in the translation of nutritional inputs into the protein synthesis machinery regulation, allowing animal growth. We recently identified the Bud32 (yeast)/PRPK (human) ortholog in Drosophila, Prpk (p53-related protein kinase), and found that it is required for TOR kinase activity. Bud32/PRPK is an ancient and atypical kinase conserved in evolution from Archeae to humans, being essential for Archeae. It has been linked with p53 stabilization in human cell culture and its absence in yeast causes a slow-growth phenotype. This protein has been associated to KEOPS (kinase, putative endopeptidase and other proteins of small size) complex together with Kae1p (ATPase), Cgi-121 and Pcc1p. This complex has been implicated in telomere maintenance, transcriptional regulation, bud site selection and chemical modification of tRNAs (tRNAs). Bud32p and Kae1p have been related with N6-threonylcarbamoyladenosine (t (6)A) synthesis, a particular chemical modification that occurs at position 37 of tRNAs that pair A-starting codons, required for proper translation in most species. Lack of this modification causes mistranslations and open reading frame shifts in yeast. The core constituents of the KEOPS complex are present in Drosophila, but their physical interaction has not been reported yet. Here, we present a review of the findings regarding the function of this complex in different organisms and new evidence that extends our recent observations of Prpk function in animal growth showing that depletion of Kae1 or Prpk, in accordance with their role in translation in yeast, is able to induce the unfolded protein response (UPR) in Drosophila. We suggest that EKC/KEOPS complex could be integrating t (6)A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth.

  8. Design and Fabrication of Nanostructures Based on DNA Ring-Protein Complex

    NASA Astrophysics Data System (ADS)

    Furukawa, Hideki; Endo, Tatsuro; Yanagida, Yasuko; Hatsuzawa, Takeshi

    2008-06-01

    In this report, we describe the design and fabrication of DNA nanostructures (“DNA glasses”, “DNA serial rings”, and “DNA chains”) using DNA ring-protein complexes. An experiment was performed to fabricate DNA ring-conjugated biotin using two types of DNA (“vector DNA” and “insert DNA”). The vector DNA was obtained by cutting plasmid DNA with restriction enzymes. The insert DNA (DNA conjugated with biotin) was obtained using a DNA synthesizer. After ligation and the introduction of streptavidin-modified gold nanoparticles, DNA structures were obtained. Subsequently, the DNA structures were observed by atomic force microscopy (AFM).

  9. Efficient Prediction of Co-Complexed Proteins Based on Coevolution

    PubMed Central

    de Vienne, Damien M.; Azé, Jérôme

    2012-01-01

    The prediction of the network of protein-protein interactions (PPI) of an organism is crucial for the understanding of biological processes and for the development of new drugs. Machine learning methods have been successfully applied to the prediction of PPI in yeast by the integration of multiple direct and indirect biological data sources. However, experimental data are not available for most organisms. We propose here an ensemble machine learning approach for the prediction of PPI that depends solely on features independent from experimental data. We developed new estimators of the coevolution between proteins and combined them in an ensemble learning procedure. We applied this method to a dataset of known co-complexed proteins in Escherichia coli and compared it to previously published methods. We show that our method allows prediction of PPI with an unprecedented precision of 95.5% for the first 200 sorted pairs of proteins compared to 28.5% on the same dataset with the previous best method. A close inspection of the best predicted pairs allowed us to detect new or recently discovered interactions between chemotactic components, the flagellar apparatus and RNA polymerase complexes in E. coli. PMID:23152796

  10. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  11. The mammalian autophagy initiator complex contains 2 HORMA domain proteins

    PubMed Central

    Michel, Max; Schwarten, Melanie; Decker, Christina; Nagel-Steger, Luitgard; Willbold, Dieter; Weiergräber, Oliver H

    2015-01-01

    ATG101 is an essential component of the ULK complex responsible for initiating cellular autophagy in mammalian cells; its 3-dimensional structure and molecular function, however, are currently unclear. Here we present the X-ray structure of human ATG101. The protein displays an open HORMA domain fold. Both structural properties and biophysical evidence indicate that ATG101 is locked in this conformation, in contrast to the prototypical HORMA domain protein MAD2. Moreover, we discuss a potential mode of dimerization with ATG13 as a fundamental aspect of ATG101 function. PMID:26236954

  12. Maize Seed Chitinase is Modified by a Protein Secreted by Bipolaris zeicola

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants contain defense mechanisms that prevent infection by most fungi. Some specialized fungi have the ability to overcome plant defenses. The Zea mays (maize) seed chitinase ChitA has been previously reported as an antifungal protein. Here we report that ChitA is converted to a modified form by...

  13. Modifying the catalytic preference of tributyrin in Bacillus thermocatenulatus lipase through in-silico modeling of enzyme-substrate complex.

    PubMed

    Durmaz, Emel; Kuyucak, Serdar; Sezerman, Ugur O

    2013-05-01

    In this study, rational design for Bacillus thermocatenulatus lipase (BTL2) was carried out to lower the activation barrier for hydrolysis of short-chain substrates. In this design, we used computational models for the enzyme-substrate (ES) complexes of tributyrin (C4) and tricaprylin (C8), which were generated through docking and molecular dynamics (MD) simulations. These ES complexes were employed in steered MD (SMD) simulations with Jarzynski's equality to estimate their relative binding free energies. Potential mutation sites for modifying the chain-length selectivity of BTL2 were found by inspecting the SMD trajectories and fine-tuning the volume and hydrophobicity of the cleft. Seven mutations (F17A, L57F, V175A, V175F, I320A, I320F and L360F) were performed to cover three binding pockets for sn-1, sn-2 and sn-3 acyl chains. The relative binding free energies of the mutant ES complexes formed by C4 and C8 ligands were calculated similarly. The experimental routines of protein engineering including site-directed mutagenesis, heterologous protein expression and purification were performed for all lipases. Steady-state specific activities towards C4 and C8 were determined for wild-type and mutant lipases, which gave an estimate of the relative change in the binding free energy of transition state complex (ES(‡)). The chain-length selectivity of mutants was determined from the relative changes in the activation barrier of hydrolysis of C4 and C8 triacylglycerol with respect to wild-type using computational and experimental findings. The most promising mutant for C4 over C8 preference was found to be L360F. We suggest that L360F may be at a critical position to lower the activation barrier for C4 and elevate it for C8 hydrolysis.

  14. Dark dyes-bright complexes: fluorogenic protein labeling.

    PubMed

    Bruchez, Marcel P

    2015-08-01

    Complexes formed between organic dyes and genetically encoded proteins combine the advantages of stable and tunable fluorescent molecules and targetable, biologically integrated labels. To overcome the challenges imposed by labeling with bright fluorescent dyes, a number of approaches now exploit chemical or environmental changes to control the properties of a bound dye, converting dyes from a weakly fluorescent state to a bright, easily detectable complex. Optimized, such approaches avoid the need for removal of unbound dyes, facilitate rapid and simple assays in cultured cells and enable hybrid labeling to function more robustly in living model organisms.

  15. Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus.

    PubMed Central

    Tagawa, Y; Haritani, M; Ishikawa, H; Yuasa, N

    1993-01-01

    In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli. Images PMID:8478064

  16. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    PubMed

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation. PMID:27390042

  17. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  18. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  19. Solid-state nanopore detection of protein complexes: applications in healthcare and protein kinetics.

    PubMed

    Freedman, Kevin J; Bastian, Arangassery R; Chaiken, Irwin; Kim, Min Jun

    2013-03-11

    Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid-state nanopores for the detection of gp120-associated complexes are investigated. They exhibit monovalent and multivalent binding to anti-gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein-specific detection schemes, which are shown to be feasible in the realm of a 'real-world' sample and an automated multidimensional method of detecting events.

  20. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    NASA Astrophysics Data System (ADS)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  1. Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces.

    PubMed

    Sun, Yan; Jallerat, Quentin; Szymanski, John M; Feinberg, Adam W

    2015-02-01

    Our Patterning on Topography (PoT) printing technique enables fibronectin, laminin and other proteins to be applied to biomaterial surfaces in complex geometries that are inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface. Here, we used this method to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment.

  2. Rational stabilization of complex proteins: a divide and combine approach

    PubMed Central

    Lamazares, Emilio; Clemente, Isabel; Bueno, Marta; Velázquez-Campoy, Adrián; Sancho, Javier

    2015-01-01

    Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones). PMID:25774740

  3. RNA-protein complexes identified by crosslinking of polysomes.

    PubMed

    Sköld, S E

    1981-01-01

    The bifunctional cleavable reagent diepoxybutane was used to investigate the crosslinking of proteins to the 16S and 23S RNA in Escherichia coli ribosomes. The crosslinking patterns from polysomes, accumulated in the absence and presence of oxytetracycline, as well as reassociated 70S ribosomes were compared. The 30S proteins: S3, S4, S5, S7, S8, S9, S12, S13, S14 and S18 were recovered crosslinked to the 16S RNA and the 50S: proteins L1, L2, L4, L13, L14-L21, L15, L16, L17, L18-L23, L19-22-24, L27 and L28 were recovered crosslinked to the 23S RNA, in all three associated states. Proteins crosslinked to the RNA of the heterologous subunit and therefore considered to be at or near the ribosomal subunit interface were, for all three states, proteins S1, S4, S6, S9, S12, S13, S14 and S18 from the small subunit and proteins L16, L17, L20 and L27 from the large subunit. Finally, the recovery of intrasubunit crosslinks was measured for the isolated subunits. Additional crosslinked complexes were observed between 16S RNA and S1, S2 as well as S6 from the 30S subunit; and between 23S RNA and L10, L11, L7/12 from the 50S subunit.

  4. De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

    PubMed

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; Ambinder, Richard F; Hayward, S Diane

    2010-11-19

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line. PMID:20847044

  5. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors*

    PubMed Central

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J.; Mobarec, Juan Carlos; Woodlock, David A.; Reynolds, Christopher A.; Poyner, David R.; Watkins, Harriet A.; Ladds, Graham

    2016-01-01

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. PMID:27566546

  6. DMS Footprinting of Structured RNAs and RNA-Protein Complexes

    PubMed Central

    Tijerina, Pilar; Mohr, Sabine; Russell, Rick

    2008-01-01

    We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nucleotides). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as ‘control’ conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis (PAGE), and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a ‘footprint’), it is possible to detect local changes in RNA secondary and tertiary structure, as well as the formation of RNA-protein contacts. This protocol takes 1.5–3 days to complete, depending on the type of analysis used. PMID:17948004

  7. Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.

    PubMed

    Eyckerman, Sven; Lemmens, Irma; Catteeuw, Dominiek; Verhee, Annick; Vandekerckhove, Joel; Lievens, Sam; Tavernier, Jan

    2005-06-01

    Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor-based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53. PMID:15908921

  8. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  9. Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking*

    PubMed Central

    Froese, D. Sean; Kopec, Jolanta; Fitzpatrick, Fiona; Schuller, Marion; McCorvie, Thomas J.; Chalk, Rod; Plessl, Tanja; Fettelschoss, Victoria; Fowler, Brian; Baumgartner, Matthias R.; Yue, Wyatt W.

    2015-01-01

    Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular “trafficking chaperone” highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations. PMID:26483544

  10. Solitary wave simulations of Complex Modified Korteweg-de Vries Equation using differential quadrature method

    NASA Astrophysics Data System (ADS)

    Korkmaz, Alper; Dağ, İdris

    2009-09-01

    Complex Modified Korteweg-deVries Equation is solved numerically using differential quadrature method based on cosine expansion. Three test problems, motion of single solitary wave, interaction of solitary waves and wave generation, are simulated. The accuracy of the method is measured via the discrete root mean square error norm L, maximum error norm L for the motion of single solitary wave since it has an analytical solution. A rate of convergency analysis for motion of single solitary wave containing both real and imaginary parts is also given. Lowest three conserved quantities are computed for all test problems. A comparison with some earlier works is given.

  11. Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

    PubMed

    Mie, Masayasu; Naoki, Tatsuhiko; Kobatake, Eiry

    2016-08-16

    The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells.

  12. Small-Angle X-Ray Scattering From RNA, Proteins, And Protein Complexes

    SciTech Connect

    Lipfert, Jan; Doniach, Sebastian; /Stanford U., Phys. Dept. /Stanford U., Appl. Phys. Dept. /SLAC, SSRL

    2007-09-18

    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

  13. Trajectory-Based Complexity (TBX): A Modified Aircraft Count to Predict Sector Complexity During Trajectory-Based Operations

    NASA Technical Reports Server (NTRS)

    Prevot, Thomas; Lee, Paul U.

    2011-01-01

    In this paper we introduce a new complexity metric to predict -in real-time- sector complexity for trajectory-based operations (TBO). TBO will be implemented in the Next Generation Air Transportation System (NextGen). Trajectory-Based Complexity (TBX) is a modified aircraft count that can easily be computed and communicated in a TBO environment based upon predictions of aircraft and weather trajectories. TBX is scaled to aircraft count and represents an alternate and additional means to manage air traffic demand and capacity with more consideration of dynamic factors such as weather, aircraft equipage or predicted separation violations, as well as static factors such as sector size. We have developed and evaluated TBX in the Airspace Operations Laboratory (AOL) at the NASA Ames Research Center during human-in-the-loop studies of trajectory-based concepts since 2009. In this paper we will describe the TBX computation in detail and present the underlying algorithm. Next, we will describe the specific TBX used in an experiment at NASA's AOL. We will evaluate the performance of this metric using data collected during a controller-inthe- loop study on trajectory-based operations at different equipage levels. In this study controllers were prompted at regular intervals to rate their current workload on a numeric scale. When comparing this real-time workload rating to the TBX values predicted for these time periods we demonstrate that TBX is a better predictor of workload than aircraft count. Furthermore we demonstrate that TBX is well suited to be used for complexity management in TBO and can easily be adjusted to future operational concepts.

  14. Atomic layer deposition modified track-etched conical nanochannels for protein sensing.

    PubMed

    Wang, Ceming; Fu, Qibin; Wang, Xinwei; Kong, Delin; Sheng, Qian; Wang, Yugang; Chen, Qiang; Xue, Jianming

    2015-08-18

    Nanopore-based devices have recently become popular tools to detect biomolecules at the single-molecule level. Unlike the long-chain nucleic acids, protein molecules are still quite challenging to detect, since the protein molecules are much smaller in size and usually travel too fast through the nanopore with poor signal-to-noise ratio of the induced transport signals. In this work, we demonstrate a new type of nanopore device based on atomic layer deposition (ALD) Al2O3 modified track-etched conical nanochannels for protein sensing. These devices show very promising properties of high protein (bovine serum albumin) capture rate with well time-resolved transport signals and excellent signal-to-noise ratio for the transport events. Also, a special mechanism involving transient process of ion redistribution inside the nanochannel is proposed to explain the unusual biphasic waveshapes of the current change induced by the protein transport.

  15. Nanospray FAIMS fractionation provides significant increases in proteome coverage of unfractionated complex protein digests.

    PubMed

    Swearingen, Kristian E; Hoopmann, Michael R; Johnson, Richard S; Saleem, Ramsey A; Aitchison, John D; Moritz, Robert L

    2012-04-01

    High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that can be used to reduce sample complexity and increase dynamic range in tandem mass spectrometry experiments. FAIMS fractionates ions in the gas-phase according to characteristic differences in mobilities in electric fields of different strengths. Undesired ion species such as solvated clusters and singly charged chemical background ions can be prevented from reaching the mass analyzer, thus decreasing chemical noise. To date, there has been limited success using the commercially available Thermo Fisher FAIMS device with both standard ESI and nanoLC-MS. We have modified a Thermo Fisher electrospray source to accommodate a fused silica pulled tip capillary column for nanospray ionization, which will enable standard laboratories access to FAIMS technology. Our modified source allows easily obtainable stable spray at flow rates of 300 nL/min when coupled with FAIMS. The modified electrospray source allows the use of sheath gas, which provides a fivefold increase in signal obtained when nanoLC is coupled to FAIMS. In this work, nanoLC-FAIMS-MS and nanoLC-MS were compared by analyzing a tryptic digest of a 1:1 mixture of SILAC-labeled haploid and diploid yeast to demonstrate the performance of nanoLC-FAIMS-MS, at different compensation voltages, for post-column fractionation of complex protein digests. The effective dynamic range more than doubled when FAIMS was used. In total, 10,377 unique stripped peptides and 1649 unique proteins with SILAC ratios were identified from the combined nanoLC-FAIMS-MS experiments, compared with 6908 unique stripped peptides and 1003 unique proteins with SILAC ratios identified from the combined nanoLC-MS experiments. This work demonstrates how a commercially available FAIMS device can be combined with nanoLC to improve proteome coverage in shotgun and targeted type proteomics experiments. PMID:22186714

  16. Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

    PubMed Central

    Shareef, Mohammed Momin; King, Chadwick; Damaj, Mona; Badagu, RamaKrishna; Huang, Da Wei; Kellum, Rebecca

    2001-01-01

    Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit. PMID:11408576

  17. Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

    PubMed Central

    Fodor, Barna D.; Kovács, Ákos T.; Csáki, Róbert; Hunyadi-Gulyás, Éva; Klement, Éva; Maróti, Gergely; Mészáros, Lívia S.; Medzihradszky, Katalin F.; Rákhely, Gábor; Kovács, Kornél L.

    2004-01-01

    A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species. PMID:14766546

  18. Modification of the protein corona-nanoparticle complex by physiological factors.

    PubMed

    Braun, Nicholas J; DeBrosse, Madeleine C; Hussain, Saber M; Comfort, Kristen K

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. PMID:27127026

  19. Modification of the protein corona-nanoparticle complex by physiological factors.

    PubMed

    Braun, Nicholas J; DeBrosse, Madeleine C; Hussain, Saber M; Comfort, Kristen K

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications.

  20. [The effect of modified nano-diamonds of detonation synthesis on the protein fractions of human blood].

    PubMed

    Botvich, Iu A; Olkhovskiĭ, I A; Baron, I I; Puzyr', A P; Baron, A V; Bondar', V S

    2013-11-01

    It is established that the modified nano-diamonds of detonation synthesis are able to bind serum proteins of human blood. The relative selectivity is established concerning the effect of modified nano-diamonds of detonation synthesis on beta2- and gamma-globulin fractions of serum. The evidence of concentration dependence of effect of modified nano-diamonds of detonation synthesis from serum proteins is established. The study results make it possible to consider modified nano-diamonds of detonation synthesis as a potential sorbent in technologies of hemodialysis, plasmapheresis, isolation of blood proteins and as a foundation for development of new systems of laboratory diagnostic.

  1. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. PMID:26519625

  2. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  3. Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions

    PubMed Central

    Tanny, Jason C.; Kirkpatrick, Donald S.; Gerber, Scott A.; Gygi, Steven P.; Moazed, Danesh

    2004-01-01

    Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo. PMID:15282295

  4. Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging.

    PubMed

    Robinson, Renã A S; Evans, Adam R

    2012-06-01

    Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy. PMID:22509719

  5. DNA-modified Electrodes Fabricated using Copper-Free Click Chemistry for Enhanced Protein Detection

    PubMed Central

    Furst, Ariel L.; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development. PMID:24328347

  6. Folding behavior of ribosomal protein S6 studied by modified Go¯ -like model

    NASA Astrophysics Data System (ADS)

    Wu, L.; Zhang, J.; Wang, J.; Li, W. F.; Wang, W.

    2007-03-01

    Recent experimental and theoretical studies suggest that, although topology is the determinant factor in protein folding, especially for small single-domain proteins, energetic factors also play an important role in the folding process. The ribosomal protein S6 has been subjected to intensive studies. A radical change of the transition state in its circular permutants has been observed, which is believed to be caused by a biased distribution of contact energies. Since the simplistic topology-only Gō -like model is not able to reproduce such an observation, we modify the model by introducing variable contact energies between residues based on their physicochemical properties. The modified Gō -like model can successfully reproduce the Φ -value distributions, folding nucleus, and folding pathways of both the wild-type and circular permutants of S6. Furthermore, by comparing the results of the modified and the simplistic models, we find that the hydrophobic effect constructs the major force that balances the loop entropies. This may indicate that nature maintains the folding cooperativity of this protein by carefully arranging the location of hydrophobic residues in the sequence. Our study reveals a strategy or mechanism used by nature to get out of the dilemma when the native structure, possibly required by biological function, conflicts with folding cooperativity. Finally, the possible relationship between such a design of nature and amyloidosis is also discussed.

  7. Potential allergenicity research of Cry1C protein from genetically modified rice.

    PubMed

    Cao, Sishuo; He, Xiaoyun; Xu, Wentao; Luo, Yunbo; Ran, Wenjun; Liang, Lixing; Dai, Yunqing; Huang, Kunlun

    2012-07-01

    With the development of genetically modified crops, there has been a growing interest in available approaches to assess the potential allergenicity of novel gene products. We were not sure whether Cry1C could induce allergy. We examined the protein with three other proteins to determine the potential allergenicity of Cry1C protein from genetically modified rice. Female Brown Norway (BN) rats received 0.1 mg peanut agglutinin (PNA), 1mg potato acid phosphatase (PAP), 1mg ovalbumin (OVA) or 5 mg purified Cry1C protein dissolved in 1 mL water by daily gavage for 42 days to test potential allergenicity. Ten days after the last gavage, rats were orally challenged with antigens, and physiologic and immunologic responses were studied. In contrast to sensitization with PNA, PAP and OVA Cry1C protein did not induce antigen-specific IgG2a in BN rats. Cytokine expression, serum IgE and histamine levels and the number of eosinophils and mast cells in the blood of Cry1C group rats were comparable to the control group rats, which were treated with water alone. As Cry1C did not show any allergenicity, we make the following conclusion that the protein could be safety used in rice or other plants.

  8. Biodegradation of the chitin-protein complex in crustacean cuticle

    USGS Publications Warehouse

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

  9. Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature[W

    PubMed Central

    Armbruster, Ute; Labs, Mathias; Pribil, Mathias; Viola, Stefania; Xu, Wenteng; Scharfenberg, Michael; Hertle, Alexander P.; Rojahn, Ulrike; Jensen, Poul Erik; Rappaport, Fabrice; Joliot, Pierre; Dörmann, Peter; Wanner, Gerhard; Leister, Dario

    2013-01-01

    Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins. PMID:23839788

  10. Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes.

    PubMed

    Kühlmann, U C; Pommer, A J; Moore, G R; James, R; Kleanthous, C

    2000-09-01

    Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme.

  11. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  12. Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

    PubMed

    Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

    2014-01-01

    In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

  13. Analytical reduction of combinatorial complexity arising from multiple protein modification sites

    PubMed Central

    Birtwistle, Marc R.

    2015-01-01

    Combinatorial complexity is a major obstacle to ordinary differential equation (ODE) modelling of biochemical networks. For example, a protein with 10 sites that can each be unphosphorylated, phosphorylated or bound to adaptor protein requires 310 ODEs. This problem is often dealt with by making ad hoc assumptions which have unclear validity and disallow modelling of site-specific dynamics. Such site-specific dynamics, however, are important in many biological systems. We show here that for a common biological situation where adaptors bind modified sites, binding is slow relative to modification/demodification, and binding to one modified site hinders binding to other sites, for a protein with n modification sites and m adaptor proteins the number of ODEs needed to simulate the site-specific dynamics of biologically relevant, lumped bound adaptor states is independent of the number of modification sites and equal to m + 1, giving a significant reduction in system size. These considerations can be relaxed considerably while retaining reasonably accurate descriptions of the true system dynamics. We apply the theory to model, using only 11 ODEs, the dynamics of ligand-induced phosphorylation of nine tyrosines on epidermal growth factor receptor (EGFR) and primary recruitment of six signalling proteins (Grb2, PI3K, PLCγ1, SHP2, RasA1 and Shc1). The model quantitatively accounts for experimentally determined site-specific phosphorylation and dephosphorylation rates, differential affinities of binding proteins for the phosphorylated sites and binding protein expression levels. Analysis suggests that local concentration of site-specific phosphatases such as SHP2 in membrane subdomains by a factor of approximately 107 is critical for effective site-specific regulation. We further show how our framework can be extended with minimal effort to consider binding cooperativity between Grb2 and c-Cbl, which is important for receptor trafficking. Our theory has potentially broad

  14. Contractile vacuole complex--its expanding protein inventory.

    PubMed

    Plattner, Helmut

    2013-01-01

    The contractile vacuole complex (CVC) of some protists serves for the osmotic equilibration of water and ions, notably Ca(2+), by chemiosmotic exploitation of a H(+) gradient generated by the organelle-resident V-type H(+)-ATPase. Ca(2+) is mostly extruded, but there is also some reflux into the cytosol via Ca(2+)-release channels. Most data available are from Dictyostelium and Paramecium. In Paramecium, the major parts of CVC contain several v-/R-SNARE (synaptobrevins) and t-/Q-SNARE (syntaxins) proteins. This is complemented by Rab-type GTPases (shown in Tetrahymena) and exocyst components (Chlamydomonas). All this reflects a multitude of membrane interactions and fusion processes. Ca(2+)/H(+) and other exchangers are to be postulated, as are aquaporins and mechanosensitive Ca(2+) channels. From the complexity of the organelle, many more proteins may be expected. For instance, the pore is endowed with its own set of proteins. We may now envisage the regulation of membrane dynamics (reversible tubulation) and the epigenetic control of organelle shape, size and positioning. New aspects about organelle function and biogenesis are sketched in Section 7. The manifold regulators currently known from CVC suggest the cooperation of widely different mechanisms to maintain its dynamic function and to drive its biogenesis.

  15. Assembly and solution structure of the core retromer protein complex.

    PubMed

    Norwood, Suzanne J; Shaw, Daniel J; Cowieson, Nathan P; Owen, David J; Teasdale, Rohan D; Collins, Brett M

    2011-01-01

    Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small-angle X-ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies. PMID:20875039

  16. Inferring drug-disease associations based on known protein complexes.

    PubMed

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  17. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  18. [Medical and biological evaluation of safety of protein concentrate from genetically-modified soybeans. Biochemical studies].

    PubMed

    Tutel'ian, V A; Kravchenko, L V; Lashneva, N V; Avren'eva, L I; Guseva, G V; Sorokina, E Iu; Chernysheva, O N

    1999-01-01

    The rats were fed with albuminous concentrate from the genetically modified soybean 40-3-2 ("Monsanto Co", USA) 1.25 g/rat/day for 5 months. Their blood, urea and liver were investigated to measure total protein and glucose levels, aminotransferase and alkaline phosphatase activities, pH, relative density and creatinine level in the urea, as well as hepatic enzyme activity of the I and II phases of xenobiotic metabolism, and the whole and non-sedimentated lysosomal enzyme activities. The lasting albuminous concentrate supplementation from the genetically modified soybean to the rat's diet has been shown to modify hepatocyte membrane function and enzymatic activity within physiological standards. It was not harmful to the adaptation systems.

  19. Young Investigator Award Lecture. Structures of larger proteins, protein-ligand and protein-DNA complexes by multidimensional heteronuclear NMR.

    PubMed Central

    Clore, G. M.; Gronenborn, A. M.

    1994-01-01

    The recent development of a whole panoply of multidimensional heteronuclear-edited and -filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the 10-kDa limit of conventional 2-dimensional NMR to systems up to potentially 35-40 kDa. The basic strategy for solving 3-dimensional structures of larger proteins and protein-ligand complexes in solution using 3- and 4-dimensional NMR spectroscopy is summarized, and the power of these methods is illustrated using 3 examples: interleukin-1 beta, the complex of calmodulin with a target peptide, and the specific complex of the transcription factor GATA-1 with its cognate DNA target site. PMID:8019409

  20. Protein complexes and functional modules in molecular networks

    NASA Astrophysics Data System (ADS)

    Spirin, Victor; Mirny, Leonid A.

    2003-10-01

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  1. Influence of the organic complex concentration on adsorption of herbicide in organic modified montmorillonite

    NASA Astrophysics Data System (ADS)

    Kaludjerovic, Lazar; Tomic, Zorica; Djurovic, Rada; Milosevic, Maja

    2016-04-01

    Pesticides are recognized as an important source of potential pollution to soil and water due to their mobility and degradation in soils. Results presented in this paper show impact of the organic complex concentration on the adsorption of herbicides (acetochlor) at the surface of the organic modified montmorillonite. In this work, natural montmorillonite from Bogovina, located near Boljevac municipality, was used for organic modification. Cation-exchange capacity of this montmorillonite was determined by extraction with ammonium acetate (86 mmol/100g of clay). Montmorillonite have been modified first with NaCl and than with two organic complexes, hexadecyltrimethylammonium bromide (HDTMA) and phenyltrimethylammonium chloride (PTMA). For both organic complexes, three saturation concentrations were selected for monitoring of the herbicide adsorption (43 mmol/100g of clay (0.5 CEC), 86 mmol/100g of clay (1 CEC) and 129 mmol/100g of clay (1.5 CEC)). Changes in the properties of the inorganic and organic bentonite have been examined using the X-ray powder diffraction (XRPD) and batch equilibrium method. Increase in basal spacing (d) of montmorillonites saturated with 1.5 CEC of organic cation indicate that sorption of PTMA and HDTMA can exceed the saturation of 1 CEC. Both organic montmorillonites have shown higher uptake of the herbicide, compared to the inorganic montmorillonite. Comparing the values Freundlich coefficients in batch equilibrium method, (presented in the form of log Kf and 1/n), it can be seen that the sorption decreases in the series: 0.5CEC> 1CEC> 1.5CEC> NaM, for both organic montmorillonites.

  2. Targeting protein–protein interactions in complexes organized by A kinase anchoring proteins

    PubMed Central

    Calejo, Ana I.; Taskén, Kjetil

    2015-01-01

    Cyclic AMP is a ubiquitous intracellular second messenger involved in the regulation of a wide variety of cellular processes, a majority of which act through the cAMP – protein kinase A (PKA) signaling pathway and involve PKA phosphorylation of specific substrates. PKA phosphorylation events are typically spatially restricted and temporally well controlled. A-kinase anchoring proteins (AKAPs) directly bind PKA and recruit it to specific subcellular loci targeting the kinase activity toward particular substrates, and thereby provide discrete spatiotemporal control of downstream phosphorylation events. AKAPs also scaffold other signaling molecules into multi-protein complexes that function as crossroads between different signaling pathways. Targeting AKAP coordinated protein complexes with high-affinity peptidomimetics or small molecules to tease apart distinct protein–protein interactions (PPIs) therefore offers important means to disrupt binding of specific components of the complex to better understand the molecular mechanisms involved in the function of individual signalosomes and their pathophysiological role. Furthermore, development of novel classes of small molecules involved in displacement of AKAP-bound signal molecules is now emerging. Here, we will focus on mechanisms for targeting PPI, disruptors that modulate downstream cAMP signaling and their role, especially in the heart. PMID:26441649

  3. GeLC-MS/MS Analysis of Complex Protein Mixtures

    PubMed Central

    Dzieciatkowska, Monika; Hill, Ryan; Hansen, Kirk C.

    2015-01-01

    Discovery-based proteomics has found its place in nearly every facet of biological research. A key objective of this approach is to maximize sequence coverage for proteins across a wide concentration range. Fractionating samples at the protein level is one of the most common ways to circumvent challenges due to sample complexity and improve proteome coverage. Of the available methods, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) is a robust and reproducible method for qualitative and quantitative proteomic analysis. Here we describe a general GeLC-MS/MS protocol and include technical advice and outline caveats to increase the probability of a successful analysis. PMID:24791981

  4. Force-induced remodelling of proteins and their complexes

    PubMed Central

    Chen, Yun; Radford, Sheena E; Brockwell, David J

    2015-01-01

    Force can drive conformational changes in proteins, as well as modulate their stability and the affinity of their complexes, allowing a mechanical input to be converted into a biochemical output. These properties have been utilised by nature and force is now recognised to be widely used at the cellular level. The effects of force on the biophysical properties of biological systems can be large and varied. As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results. Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study. PMID:25710390

  5. Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

    PubMed

    Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

    2016-06-01

    Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

  6. Retention of Proteins and Metalloproteins in Open Tubular Capillary Electrochromatography with Etched Chemically Modified Columns

    PubMed Central

    Pesek, Joseph J.; Matyska, Maria T.; Salgotra, Vasudha

    2010-01-01

    Etched chemically modified capillaries with two different bonded groups (pentyl and octadecyl) are compared for their migration behavior of several common proteins and metalloproteins as well as metalloproteinases. Migration times, efficiency and peak shape are evaluated over the pH range of 2.1 to 8.1 to determine any effects of the bonded group on the electrochromatographic behavior of these compounds. One goal was to determine if the relative hydrophobicity of the stationary phase has a significant effect on proteins in the open tubular format of capillary electrochromatography as it does in HPLC. Reproducibility of the migration times is also investigated. PMID:18850653

  7. [The investigation of presence of genetically modified protein in processed foodstuffs by ELISA test].

    PubMed

    Urbanek-Karłowska, Bogumiła; Sawilska-Rautenstrauch, Dorota; Jedra, Małgorzata

    2004-01-01

    The test based on immunoassay--TRAIT-RUR Toasted Soy Meal Kit was used for detection GMO-soy protein in the processed (heat treated) foodstuffs: bread, macaroni, sausages, ready-to-serve products and soya products (tofu, steaks). The threshold level is about 0,6% protein. The positive results were obtained for 27 from 106 investigated products. Only 5 foodstuffs were declared as containing GMO-soy in composition. The presence of genetically modified ingredients in foodstuffs must be controlled. The proper information should be labelled for the consumer.

  8. Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

    PubMed

    Kamath, Padmaja; Fernandez, Alberto; Giralt, Francesc; Rallo, Robert

    2015-01-01

    Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona.

  9. Computational allergenicity prediction of transgenic proteins expressed in genetically modified crops.

    PubMed

    Verma, Alok Kumar; Misra, Amita; Subash, Swarna; Das, Mukul; Dwivedi, Premendra D

    2011-09-01

    Development of genetically modified (GM) crops is on increase to improve food quality, increase harvest yields, and reduce the dependency on chemical pesticides. Before their release in marketplace, they should be scrutinized for their safety. Several guidelines of different regulatory agencies like ILSI, WHO Codex, OECD, and so on for allergenicity evaluation of transgenics are available and sequence homology analysis is the first test to determine the allergenic potential of inserted proteins. Therefore, to test and validate, 312 allergenic, 100 non-allergenic, and 48 inserted proteins were assessed for sequence similarity using 8-mer, 80-mer, and full FASTA search. On performing sequence homology studies, ~94% the allergenic proteins gave exact matches for 8-mer and 80-mer homology. However, 20 allergenic proteins showed non-allergenic behavior. Out of 100 non-allergenic proteins, seven qualified as allergens. None of the inserted proteins demonstrated allergenic behavior. In order to improve the predictability, proteins showing anomalous behavior were tested by Algpred and ADFS separately. Use of Algpred and ADFS softwares reduced the tendency of false prediction to a great extent (74-78%). In conclusion, routine sequence homology needs to be coupled with some other bioinformatic method like ADFS/Algpred to reduce false allergenicity prediction of novel proteins.

  10. The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins.

    PubMed

    Evans, T C; Benner, J; Xu, M Q

    1999-06-25

    Mini-inteins derived from Synechocystis sp. (Ssp DnaB intein) and Mycobacterium xenopi (Mxe GyrA intein) that have been modified to cleave peptide bonds at their C and N termini, respectively, were cloned in-frame to the N and C termini of a target protein. Peptide bond cleavage of the modified inteins generated an N-terminal cysteine and a C-terminal thioester on the same protein. These complementary reactive groups underwent intra- or intermolecular condensation to generate circular or polymeric protein species with a new peptide bond at the site of ligation. Three cyclic peptides, BBP, an organ specific localization peptide; RGD, an inhibitor of platelet aggregation; and CDR-H3/C2, which inhibits HIV-1 replication, were isolated using the two-intein system. BBP, RGD, and CDR-H3/C2 had masses of 977.1, 1119.9, and 2098.6 g/mol, respectively, as determined by matrix-assisted laser desorption-time of flight mass spectrometry, which agreed well with the values of 977.2, 1120.3, and 2098.3 g/mol, respectively, predicted for the cyclic species. This system was used to cyclize proteins as large as 395 amino acids. Furthermore, multimers of thioredoxin were formed upon concentration of the reactive species, indicating the potential to form novel biomaterials based on fibrous proteins.

  11. G-protein Coupled Receptor 30 Interacts with Receptor Activity Modifying Protein 3 and Confers Sex-Dependent Cardioprotection

    PubMed Central

    Lenhart, Patricia M.; Broselid, Stefan; Barrick, Cordelia J.; Leeb-Lundberg, L.M. Fredrik; Caron, Kathleen M.

    2013-01-01

    Receptor activity modifying protein 3 (RAMP3) is a single pass transmembrane protein known to interact with and affect the trafficking of several G-protein coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with G-protein coupled receptor 30 (GPR30), also known as G-protein estrogen receptor 1 (GPER1). GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Utilizing bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3- and sex-dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo, and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  12. Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin.

    PubMed

    Masuda, Y; Nirasawa, S; Nakaya, K; Kurihara, Y

    1995-08-19

    A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and was present specifically in the pulp. PMID:7665074

  13. Integration of genomic datasets to predict protein complexes in yeast.

    PubMed

    Jansen, Ronald; Lan, Ning; Qian, Jiang; Gerstein, Mark

    2002-01-01

    The ultimate goal of functional genomics is to define the function of all the genes in the genome of an organism. A large body of information of the biological roles of genes has been accumulated and aggregated in the past decades of research, both from traditional experiments detailing the role of individual genes and proteins, and from newer experimental strategies that aim to characterize gene function on a genomic scale. It is clear that the goal of functional genomics can only be achieved by integrating information and data sources from the variety of these different experiments. Integration of different data is thus an important challenge for bioinformatics. The integration of different data sources often helps to uncover non-obvious relationships between genes, but there are also two further benefits. First, it is likely that whenever information from multiple independent sources agrees, it should be more valid and reliable. Secondly, by looking at the union of multiple sources, one can cover larger parts of the genome. This is obvious for integrating results from multiple single gene or protein experiments, but also necessary for many of the results from genome-wide experiments since they are often confined to certain (although sizable) subsets of the genome. In this paper, we explore an example of such a data integration procedure. We focus on the prediction of membership in protein complexes for individual genes. For this, we recruit six different data sources that include expression profiles, interaction data, essentiality and localization information. Each of these data sources individually contains some weakly predictive information with respect to protein complexes, but we show how this prediction can be improved by combining all of them. Supplementary information is available at http:// bioinfo.mbb.yale.edu/integrate/interactions/. PMID:12836664

  14. The Complex Energy Landscape of the Protein IscU.

    PubMed

    Bothe, Jameson R; Tonelli, Marco; Ali, Ibrahim K; Dai, Ziqi; Frederick, Ronnie O; Westler, William M; Markley, John L

    2015-09-01

    IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar (1)H-(15)N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU.

  15. The Complex Energy Landscape of the Protein IscU

    PubMed Central

    Bothe, Jameson R.; Tonelli, Marco; Ali, Ibrahim K.; Dai, Ziqi; Frederick, Ronnie O.; Westler, William M.; Markley, John L.

    2015-01-01

    IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar 1H-15N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU. PMID:26331259

  16. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  17. Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

    PubMed

    Eliahoo, Elad; Litovco, Phyana; Ben Yosef, Ron; Bendalak, Keren; Ziv, Tamar; Manor, Haim

    2014-04-01

    Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

  18. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    PubMed

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs. PMID:27499021

  19. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  20. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    PubMed

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

  1. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

    PubMed Central

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  2. Estimation of complex Young's modulus of non-stiff materials using a modified Oberst beam technique

    NASA Astrophysics Data System (ADS)

    Liao, Yabin; Wells, Valana

    2008-09-01

    The paper presents a modified Oberst beam technique to evaluate the complex Young's modulus of non-stiff materials. Unconstrained layer theories and non-parametric complex modulus identification methods used for stiff materials form the basis for the method. The proposed approach has several advantages over the standard Oberst beam technique. In particular, the layer properties can be evaluated at any frequency, and the base beam need not be completely covered with the layer material. In addition, the proposed method does not require that the complex modulus vs. frequency curve for the base beam should have a flat area near analyzed resonance frequencies in order to yield accurate results. The experiments conducted on a styrene-butadiene rubber (SBR) sample using a polymethyl methacrylate (PMMA) base beam produced good results. Uncertainty analysis shows that the measurement accuracy can be improved by increasing the modulus magnitude ratio between the layer material and the base beam material, or the thickness ratio between the layer and base beam.

  3. Cerium(III) Complex Modified Gold Electrode: An Efficient Electrocatalyst for the Oxygen Evolution Reaction.

    PubMed

    Garain, Samiran; Barman, Koushik; Sinha, Tridib Kumar; Jasimuddin, Sk; Haeberle, Jörg; Henkel, Karsten; Schmeisser, Dieter; Mandal, Dipankar

    2016-08-24

    Exploring efficient and inexpensive electrocatalysts for the oxidation of water is of great importance for various electrochemical energy storage and conversion technologies. In the present study, a new water-soluble [Ce(III)(DMF) (HSO4)3] complex was synthesized and characterized by UV-vis, photoluminescence, and high-resolution X-ray photoelectron spectroscopy techniques. Owing to classic 5d → 4f transitions, an intense photoluminescence in the UV region was observed from the water-soluble [Ce(III)(DMF) (HSO4)3] complex. A stacking electrode was designed where self-assembled l-cysteine monolayer modified gold was immobilized with the synthesized cerium complex and was characterized by scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. The resulting electrode, i.e., [Ce(III)(DMF) (HSO4)3]-l-cysteine-Au stacks shows high electrocatalytic water oxidation behavior at an overpotential of η ≈ 0.34 V under neutral pH conditions. We also demonstrated a way where the overpotential is possible to decrease upon irradiation of UV light. PMID:27490440

  4. Protein adsorption to poly(ethylenimine)-modified Sepharose FF: III. Comparison between different proteins.

    PubMed

    Hong, Yan; Liu, Na; Wei, Wei; Yu, Lin-Ling; Ma, Guanghui; Sun, Yan

    2014-05-16

    Previously, we studied bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose resins, and an ionic capacity (IC) range (600-740mmol/L) for steep increases of both protein capacity (qm) and effective pore diffusion coefficient (De) was found. In this work, seven PEI-grafted Sepharose FF resins at IC range of 270-1030mmol/L were synthesized to investigate the effect of protein properties on the adsorption and uptake kinetics using BSA and γ-globulin as two model proteins. For BSA, the change trends of qm and De values with IC were well consistent with the previous results. For γ-globulin, the qm values increased slowly till reaching a maximum value at IC=560mmol/L and then decreased rapidly at IC>560mol/L. The De values nearly kept unchanged at low ICs (IC<460mmol/L), and increased steeply at IC>460mmol/L till reaching a maximum at 680mmol/L (De/D0=0.48±0.01). After that increase, the De values for γ-globulin dropped quickly at IC>680mol/L, which was not observed for BSA. It is interesting to note that in the narrow IC range of 460-680mmol/L, the De values of γ-globulin increased dramatically for more than four folds. Moreover, it is notable that the IC range where the hopping of De values occurred for γ-globulin was earlier than that for BSA (460 vs. 560mmol/L). The earlier hopping of γ-globulin uptake rate was attributed to its larger size and less net charge, which facilitated the happenings of the "chain delivery" effect. The quick drops of both qm and De values for γ-globulin at IC>680mmol/L were considered due to its large size, which led to the significant decrease of its effective pore volume. The results indicate that both PEI layer and protein size played important roles in protein adsorption to PEI-grafted resins, and further prove the "chain delivery" effect did contributed significantly to the uptake rate hopping in the PEI-grafted resins. This work could also help the design and selection of resins based on protein

  5. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  6. Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells.

    PubMed

    Hirai, Tadayoshi; Sato, Mayuko; Toyooka, Kiminari; Sun, Hyeon-Jin; Yano, Megumu; Ezura, Hiroshi

    2010-02-15

    A taste-modifying protein, miraculin, is highly accumulated in ripe fruit of miracle fruit (Richadella dulcifica) and the content can reach up to 10% of the total soluble protein in these fruits. Although speculated for decades that miraculin is secreted into intercellular spaces in miracle fruit, no evidence exists of its cellular localization. To study the cellular localization of miraculin in plant cells, using miracle fruit and transgenic tomato that constitutively express miraculin, immunoelectron microscopy, imaging GFP fusion proteins, and immunological detection of secreted proteins in culture medium of transgenic tomato were carried out. Immunoelectron microscopy showed the specific accumulation of miraculin in the intercellular layers of both miracle fruit and transgenic tomato. Imaging GFP fusion protein demonstrated that the miraculin-GFP fusion protein was accumulated in the intercellular spaces of tomato epidermal cells. Immunological detection of secreted proteins in culture medium of transgenic tomato indicated that miraculin was secreted from the roots of transgenic tomato expressing miraculin. This study firstly showed the evidences of the intercellular localization of miraculin, and provided a new insight of biological roles of miraculin in plants. PMID:19712996

  7. An efficient modified Gabriel method for triangulating complex fractured media for multiphase flow simulations

    NASA Astrophysics Data System (ADS)

    Mustapha, Hussein; Rouxel-Labbé, Mael; Abbas, Hicham

    2013-10-01

    Fractured reservoirs and aquifers are complex domains where discrete fractures are internal constraining boundaries. The Delaunay triangulation of a fractured medium generally does not conform to the fracture boundaries and recovering the fracture elements may violate the Delaunay empty-circle (2D) criterion, which may lead to a low-quality triangulation. This paper presents a new approach based on the combined Gabriel and Delaunay methods. A modified Gabriel condition of edge-empty-circle is introduced. In a first stage, the fracture edges violating the modified Gabriel criterion are released and then followed by a Delaunay triangulation with the rest of the fracture constraints. The released fracture edges are approximated by the edges of the Delaunay triangles in a postprocessing stage. The final representation of the fractures might be slightly different, but a very accurate solution is always maintained. The method has the capability to generate fine grids and to offer an accurate and good-quality grid. Numerical examples are presented to assess the efficiency of the proposed method.

  8. Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells.

    PubMed

    Aekbote, Badri L; Fekete, Tamás; Jacak, Jaroslaw; Vizsnyiczai, Gaszton; Ormos, Pál; Kelemen, Lóránd

    2016-01-01

    We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and non-specifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation. PMID:26819816

  9. Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells

    PubMed Central

    Aekbote, Badri L.; Fekete, Tamás; Jacak, Jaroslaw; Vizsnyiczai, Gaszton; Ormos, Pál; Kelemen, Lóránd

    2015-01-01

    We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and non-specifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation. PMID:26819816

  10. G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection.

    PubMed

    Lenhart, Patricia M; Broselid, Stefan; Barrick, Cordelia J; Leeb-Lundberg, L M Fredrik; Caron, Kathleen M

    2013-01-01

    Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3(-/-) mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3(+)(/)(+) and Ramp3(-/-) mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  11. Determination of protein-ligand interactions using accelerator mass spectrometry: modified crosslinking assay.

    PubMed

    Hah, Sang Soo

    2009-05-01

    A highly sensitive detection method for the determination of protein-ligand interactions has been developed. Radiocarbon-labeled 17beta-estradiol was incubated with estrogen receptor-alpha; as a selective binding partner, and covalently attached using crosslinking agents, to form covalently linked protein-ligand complexes. After separation using a denaturing gel, the (14)C content in the sliced gels was identified by accelerator mass spectrometry. The obtained data demonstrated specific binding of the small molecule to its binding partner. In theory, this method can be applied to most protein-ligand interaction studies.

  12. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

    PubMed Central

    Davies, Douglas R.; Gelinas, Amy D.; Zhang, Chi; Rohloff, John C.; Carter, Jeffrey D.; O’Connell, Daniel; Waugh, Sheela M.; Wolk, Steven K.; Mayfield, Wesley S.; Burgin, Alex B.; Edwards, Thomas E.; Stewart, Lance J.; Gold, Larry; Janjic, Nebojsa; Jarvis, Thale C.

    2012-01-01

    Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets. PMID:23139410

  13. Pseudomonas aeruginosa PA1006 Is a Persulfide-Modified Protein That Is Critical for Molybdenum Homeostasis

    PubMed Central

    Tombline, Gregory; Schwingel, Johanna M.; Lapek, John D.; Friedman, Alan E.; Darrah, Thomas; Maguire, Michael; Van Alst, Nadine E.; Filiatrault, Melanie J.; Iglewski, Barbara H.

    2013-01-01

    A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) “top-down” analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein’s persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant. PMID:23409003

  14. CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection.

    PubMed

    Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

    2007-07-01

    The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.

  15. A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses*

    PubMed Central

    Song, Xuezheng; Yu, Hai; Chen, Xi; Lasanajak, Yi; Tappert, Mary M.; Air, Gillian M.; Tiwari, Vinod K.; Cao, Hongzhi; Chokhawala, Harshal A.; Zheng, Haojie; Cummings, Richard D.; Smith, David F.

    2011-01-01

    Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2–3- and α2–6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2–6- and α2–3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-d-glycero-d-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2–6-linked Neu5Ac9Lt or α2–6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions. PMID:21757734

  16. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    PubMed Central

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof , Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif , Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  17. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.

    PubMed

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof, Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif, Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  18. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

    PubMed

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S; Cuéllar Pérez, Amparo; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-08-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  19. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  20. Hierarchical structures made of proteins. The complex architecture of spider webs and their constituent silk proteins.

    PubMed

    Heim, Markus; Römer, Lin; Scheibel, Thomas

    2010-01-01

    Biopolymers fulfil a variety of different functions in nature. They conduct various processes inside and outside cells and organisms, with a functionality ranging from storage of information to stabilization, protection, shaping, transport, cellular division, or movement of whole organisms. Within the plethora of biopolymers, the most sophisticated group is of proteinaceous origin: the cytoskeleton of a cell is made of protein filaments that aid in pivotal processes like intracellular transport, movement, and cell division; geckos use a distinct arrangement of keratin-like filaments on their toes which enable them to walk up smooth surfaces, such as walls, and even upside down across ceilings; and spiders spin silks that are extra-corporally used for protection of offspring and construction of complex prey traps. The following tutorial review describes the hierarchical organization of protein fibers, using spider dragline silk as an example. The properties of a dragline silk thread originate from the strictly controlled assembly of the underlying protein chains. The assembly procedure leads to protein fibers showing a complex hierarchical organization comprising three different structural phases. This structural organization is responsible for the outstanding mechanical properties of individual fibers, which out-compete even those of high-performance artificial fibers like Kevlar. Web-weaving spiders produce, in addition to dragline silk, other silks with distinct properties, based on slightly variant constituent proteins--a feature that allows construction of highly sophisticated spider webs with well designed architectures and with optimal mechanical properties for catching prey.

  1. A pearl protein self-assembles to form protein complexes that amplify mineralization.

    PubMed

    Perovic, Iva; Mandal, Trinanjana; Evans, John Spencer

    2013-08-20

    The formation of the nacre pearl in marine invertebrates represents an on-demand production of mineralization in response to an irritant or parasite threat to the mantle organ. In the Japanese pearl oyster (Pinctada fucata), this process is mediated by a 12-member protein family known as PFMG (Pinctada fucata mantle gene). One of these proteins, PFGM1, has been implicated in modulating calcium carbonate crystal growth and has been reported to possess an EF-hand-like domain. In this report, we establish that the recombinant PFMG1 (rPFMG1) is an intrinsically disordered "imitator" EF-hand protein that increases the number of calcium carbonate mineral crystals that form relative to control scenarios and does not induce aragonite formation. This protein possesses a modified pseudo-EF-hand sequence at the C-terminal end which exhibits low homology (30-40%) to the pseudo-EF-hand mitochondrial SCaMCs buffering/solute transport proteins. This low sequence homology is the result of the inclusion of disorder-promoting amino acids and short amyloid-like aggregation-prone cross-β-strand sequences within the putative PFMG1 pseudo-EF-hand sequence region. Similar to other nacre proteins, rPFMG1 oligomerizes to form amorphous, heterogeneously sized protein oligomers and films in vitro, and this process is enhanced by Ca(2+), which promotes the formation of aggregation-prone extended β-strand structure within rPFMG1. From these results, we conclude that PFMG1 forms supramolecular assemblies that play an important role in amplifying the nucleation process that is crucial for coating or neutralizing invasive threats to the mantle organ. PMID:23865482

  2. A pearl protein self-assembles to form protein complexes that amplify mineralization.

    PubMed

    Perovic, Iva; Mandal, Trinanjana; Evans, John Spencer

    2013-08-20

    The formation of the nacre pearl in marine invertebrates represents an on-demand production of mineralization in response to an irritant or parasite threat to the mantle organ. In the Japanese pearl oyster (Pinctada fucata), this process is mediated by a 12-member protein family known as PFMG (Pinctada fucata mantle gene). One of these proteins, PFGM1, has been implicated in modulating calcium carbonate crystal growth and has been reported to possess an EF-hand-like domain. In this report, we establish that the recombinant PFMG1 (rPFMG1) is an intrinsically disordered "imitator" EF-hand protein that increases the number of calcium carbonate mineral crystals that form relative to control scenarios and does not induce aragonite formation. This protein possesses a modified pseudo-EF-hand sequence at the C-terminal end which exhibits low homology (30-40%) to the pseudo-EF-hand mitochondrial SCaMCs buffering/solute transport proteins. This low sequence homology is the result of the inclusion of disorder-promoting amino acids and short amyloid-like aggregation-prone cross-β-strand sequences within the putative PFMG1 pseudo-EF-hand sequence region. Similar to other nacre proteins, rPFMG1 oligomerizes to form amorphous, heterogeneously sized protein oligomers and films in vitro, and this process is enhanced by Ca(2+), which promotes the formation of aggregation-prone extended β-strand structure within rPFMG1. From these results, we conclude that PFMG1 forms supramolecular assemblies that play an important role in amplifying the nucleation process that is crucial for coating or neutralizing invasive threats to the mantle organ.

  3. Electrochemically modified carbon and chromium surfaces for AFM imaging of double-strand DNA interaction with transposase protein.

    PubMed

    Esnault, Charles; Chénais, Benoît; Casse, Nathalie; Delorme, Nicolas; Louarn, Guy; Pilard, Jean-François

    2013-02-01

    Carbon and chromium surfaces were modified by electrochemical reduction of a diazonium salt formed in situ from the sulfanilic acid. The organic layer formed was activated by phosphorus pentachloride (PCl(5)) to form a benzene sulfonil chloride (Ar-SO(2)Cl). An electrochemical study of the blocking effect and the activity of this surface was carried out on a carbon electrode. The chromium surface study was completed by X-ray photoelectron spectroscopy and atomic force microscopy to characterize the formation of a compact monolayer (0.8 nm height and roughness 0.2-0.3 nm). The compactness and the activity of this organic monolayer allowed us to affix a length dsDNA with the aim of analyzing the formation of a complex between dsDNA and a protein. The interaction of a transposase protein with its target dsDNA was investigated. The direct imaging of the nucleoproteic complex considered herein gives new insights in the comprehension of transposase-DNA interaction in agreement with biochemical data.

  4. Controlled release of NELL-1 protein from chitosan/hydroxyapatite-modified TCP particles.

    PubMed

    Zhang, Yulong; Dong, Rui; Park, Yujin; Bohner, Marc; Zhang, Xinli; Ting, Kang; Soo, Chia; Wu, Benjamin M

    2016-09-10

    NEL-like molecule-1 (NELL-1) is a novel osteogenic protein that showing high specificity to osteochondral cells. It was widely used in bone regeneration research by loading onto carriers such as tricalcium phosphate (TCP) particles. However, there has been little research on protein controlled release from this material and its potential application. In this study, TCP was first modified with a hydroxyapatite coating followed by a chitosan coating to prepare chitosan/hydroxyapatite-coated TCP particles (Chi/HA-TCP). The preparation was characterized by SEM, EDX, FTIR, XRD, FM and Zeta potential measurements. The NELL-1 loaded Chi/HA-TCP particles and the release kinetics were investigated in vitro. It was observed that the Chi/HA-TCP particles prepared with the 0.3% (wt/wt) chitosan solution were able to successfully control the release of NELL-1 and maintain a slow, steady release for up to 28 days. Furthermore, more than 78% of the loaded protein's bioactivity was preserved in Chi/HA-TCP particles over the period of the investigation, which was significantly higher than that of the protein released from hydroxyapatite coated TCP (HA-TCP) particles. Collectively, this study suggests that the osteogenic protein NELL-1 showed a sustained release pattern after being encapsulated into the modified Chi/HA-TCP particles, and the NELL-1 integrated composite of Chi/HA-TCP showed a potential to function as a protein delivery carrier and as an improved bone matrix for use in bone regeneration research. PMID:27349789

  5. Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex.

    PubMed

    Dyall, Sabrina D; Yan, Weihong; Delgadillo-Correa, Maria G; Lunceford, Adam; Loo, Joseph A; Clarke, Catherine F; Johnson, Patricia J

    2004-10-28

    Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont.

  6. Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex.

    PubMed

    Dyall, Sabrina D; Yan, Weihong; Delgadillo-Correa, Maria G; Lunceford, Adam; Loo, Joseph A; Clarke, Catherine F; Johnson, Patricia J

    2004-10-28

    Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont. PMID:15510149

  7. Identifying dynamic protein complexes based on gene expression profiles and PPI networks.

    PubMed

    Li, Min; Chen, Weijie; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2014-01-01

    Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of "closeness" and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

  8. Molecular mechanisms of the action of miraculin, a taste-modifying protein.

    PubMed

    Misaka, Takumi

    2013-03-01

    Miraculin (MCL) is a homodimeric protein isolated from the fruits of Richadella dulcifica, a shrub native to West Africa. Although it is flat in taste at neutral pH, MCL has taste-modifying activity in which sour stimuli produce a sweet perception. Once MCL enters the mouth, strong sweetness can be detected for more than 1 h each time we taste a sour solution. While the human sweet taste receptor (hT1R2-hT1R3) has been identified, the molecular mechanisms underlying the taste-modifying activity of MCL remain unclear. Recently, experimental evidence has been published demonstrating the successful quantitative evaluation of the acid-induced sweetness of MCL using a cell-based assay system. The results strongly suggested that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH. Since sweet-tasting proteins may be used as low-calorie sweeteners because they contain almost no calories, it is expected that MCL will be used in the near future as a new low-calorie sweetener or to modify the taste of sour fruits. PMID:23466289

  9. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  10. ML3 Is a NEDD8- and Ubiquitin-Modified Protein1[C][W][OPEN

    PubMed Central

    Hakenjos, Jana P.; Bejai, Sarosh; Ranftl, Quirin; Behringer, Carina; Vlot, A. Corina; Absmanner, Birgit; Hammes, Ulrich; Heinzlmeir, Stephanie; Kuster, Bernhard; Schwechheimer, Claus

    2013-01-01

    NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED PROTEIN8) is an evolutionarily conserved 8-kD protein that is closely related to ubiquitin and that can be conjugated like ubiquitin to specific lysine residues of target proteins in eukaryotes. In contrast to ubiquitin, for which a broad range of substrate proteins are known, only a very limited number of NEDD8 target proteins have been identified to date. Best understood, and also evolutionarily conserved, is the NEDD8 modification (neddylation) of cullins, core subunits of the cullin-RING-type E3 ubiquitin ligases that promote the polyubiquitylation of degradation targets in eukaryotes. Here, we show that Myeloid differentiation factor-2-related lipid-recognition domain protein ML3 is an NEDD8- as well as ubiquitin-modified protein in Arabidopsis (Arabidopsis thaliana) and examine the functional role of ML3 in the plant cell. Our analysis indicates that ML3 resides in the vacuole as well as in endoplasmic reticulum (ER) bodies. ER bodies are Brassicales-specific ER-derived organelles and, similar to other ER body proteins, ML3 orthologs can only be identified in this order of flowering plants. ML3 gene expression is promoted by wounding as well as by the phytohormone jasmonic acid and repressed by ethylene, signals that are known to induce and repress ER body formation, respectively. Furthermore, ML3 protein abundance is dependent on NAI1, a master regulator of ER body formation in Arabidopsis. The regulation of ML3 expression and the localization of ML3 in ER bodies and the vacuole is in agreement with a demonstrated importance of ML3 in the defense to herbivore attack. Here, we extend the spectrum of ML3 biological functions by demonstrating a role in the response to microbial pathogens. PMID:23903439

  11. [Studies on protein-based identification method of genetically modified capsicum].

    PubMed

    Liu, Jianjun; Deng, Pingjian; Fang, Shisong; Zhao, Jin

    2003-03-01

    The detection system based on protein is a method to evaluate the safety of genetically modified foods (GMF). Using cecropin BD gene in capsicum, a detecting method was set up. It is a system of evaluating the real expressive condition and safety of the foreign target protein of GMF. In this studies, with the preformative technic method, a satisfactory results by making use of hemolymph of immunized pupae of Antheraea pernyi as standard experimental material was achieved, comparing with the realities of the goal protein expressive condition of cecropin D gene in capsicum. The detecting steps were as following: the goal protein from material was extracted roughly, then with CM-Sepharose-FF ion-exchange chromatography twice, the goal protein was purified moderately. The purified product was identified by detecting the anti-bacterial activity, electrophoresis, biological auto-photography of the goal protein and MADDI-TOF mass spectrum. The results showed that the expressive foreign target protein in transgenic capsicum was in accordance with standard protein in the physical and chemical property, anti-bacterial activity and molecular weight. It indicated that expression of the target gene in capsicum is real, it corresponded to expected value. The separation, purification and identification methods of cecropin D were established in the study. By means of the comparative experiments about anti-bacterial activity and molecular weight of anti-bacterial peptide(ABP) from GM-capsicum and hemolymph of immunized pupae of Antheraea pernyi, the identification method of target protein from GM-capsicum was set up. The method is easy to be operated, fast and feasible.

  12. Docking and scoring protein complexes: CAPRI 3rd Edition.

    PubMed

    Lensink, Marc F; Méndez, Raúl; Wodak, Shoshana J

    2007-12-01

    The performance of methods for predicting protein-protein interactions at the atomic scale is assessed by evaluating blind predictions performed during 2005-2007 as part of Rounds 6-12 of the community-wide experiment on Critical Assessment of PRedicted Interactions (CAPRI). These Rounds also included a new scoring experiment, where a larger set of models contributed by the predictors was made available to groups developing scoring functions. These groups scored the uploaded set and submitted their own best models for assessment. The structures of nine protein complexes including one homodimer were used as targets. These targets represent biologically relevant interactions involved in gene expression, signal transduction, RNA, or protein processing and membrane maintenance. For all the targets except one, predictions started from the experimentally determined structures of the free (unbound) components or from models derived by homology, making it mandatory for docking methods to model the conformational changes that often accompany association. In total, 63 groups and eight automatic servers, a substantial increase from previous years, submitted docking predictions, of which 1994 were evaluated here. Fifteen groups submitted 305 models for five targets in the scoring experiment. Assessment of the predictions reveals that 31 different groups produced models of acceptable and medium accuracy-but only one high accuracy submission-for all the targets, except the homodimer. In the latter, none of the docking procedures reproduced the large conformational adjustment required for correct assembly, underscoring yet again that handling protein flexibility remains a major challenge. In the scoring experiment, a large fraction of the groups attained the set goal of singling out the correct association modes from incorrect solutions in the limited ensembles of contributed models. But in general they seemed unable to identify the best models, indicating that current scoring

  13. Surface hydrophobization by electrostatic deposition of hydrophobically modified poly(acrylates) and their complexes with surfactants

    NASA Astrophysics Data System (ADS)

    Gîfu, Ioana Cătălina; Maxim, Monica Elisabeta; Iovescu, Alina; Simion, Elena Livia; Aricov, Ludmila; Anastasescu, Mihai; Munteanu, Cornel; Anghel, Dan-Florin

    2016-05-01

    The present study demonstrates the hydrophobic effect of poly(electrolyte) multilayer films when they are alkyl-grafted and complexed or not with surfactants. For this purpose, sodium hydrophobically modified poly(acrylates) (PACnNa, n = 10, 18) or their anionic complexes with alkyltrimethylammonium bromides (CxTAB, x = 10, 12, 14, 18), and the cationic poly(diallyldimethyldiammonium chloride) (PDDAMAC) are assembled by layer-by-layer deposition on a glass substrate. Contact angle (CA) measurements reveal that films constructed with PACnNa-CxTAB/PDADMAC are superior water repellants than those of PACnNa/PDADMAC. For example, the highest CA is obtained for the PAC18Na-C18TAB/PDADMAC. Moreover, it has been observed that the CA increases with the alkyl chain length of PACnNa and of surfactant. The film roughness and thickness have the same trend as wettability. Thinner and less coarse films are obtained by NaCl addition, as witnessed by SEM and AFM.

  14. CCM1 and the second life of proteins in adhesion complexes

    PubMed Central

    van den Berg, Maaike CW; Burgering, Boudewijn MT

    2014-01-01

    It is well recognized that a number of proteins present within adhesion complexes perform discrete signaling functions outside these adhesion complexes, including transcriptional control. In this respect, β-catenin is a well-known example of an adhesion protein present both in cadherin complexes and in the nucleus where it regulates the TCF transcription factor. Here we discuss nuclear functions of adhesion complex proteins with a special focus on the CCM-1/KRIT-1 protein, which may turn out to be yet another adhesion complex protein with a second life. PMID:24714220

  15. Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

    PubMed

    Moor, Nina A; Vasil'eva, Inna A; Anarbaev, Rashid O; Antson, Alfred A; Lavrik, Olga I

    2015-07-13

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

  16. Heterodimeric protein complex identification by naïve Bayes classifiers

    PubMed Central

    2013-01-01

    Background Protein complexes are basic cellular entities that carry out the functions of their components. It can be found that in databases of protein complexes of yeast like CYC2008, the major type of known protein complexes is heterodimeric complexes. Although a number of methods for trying to predict sets of proteins that form arbitrary types of protein complexes simultaneously have been proposed, it can be found that they often fail to predict heterodimeric complexes. Results In this paper, we have designed several features characterizing heterodimeric protein complexes based on genomic data sets, and proposed a supervised-learning method for the prediction of heterodimeric protein complexes. This method learns the parameters of the features, which are embedded in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve Bayes classifier with the parameter values obtained by maximum likelihood estimation gives the score of a given pair of proteins to predict whether the pair is a heterodimeric complex or not. A five-fold cross-validation shows good performance on yeast. The trained classifiers also show higher predictability than various existing algorithms on yeast data sets with approximate and exact matching criteria. Conclusions Heterodimeric protein complex prediction is a rather harder problem than heteromeric protein complex prediction because heterodimeric protein complex is topologically simpler. However, it turns out that by designing features specialized for heterodimeric protein complexes, predictability of them can be improved. Thus, the design of more sophisticate features for heterodimeric protein complexes as well as the accumulation of more accurate and useful genome-wide data sets will lead to higher predictability of heterodimeric protein complexes. Our tool can be downloaded from http://imi.kyushu-u.ac.jp/~om/. PMID:24299017

  17. Quantification of Poly(ADP-ribose)-Modified Proteins in Cerebrospinal Fluid from Infants and Children after Traumatic Brain Injury

    PubMed Central

    Fink, Ericka L.; Lai, Yichen; Zhang, Xiaopeng; Janesko-Feldman, Keri; Adelson, P. David; Szabó, Csaba; Berger, Rachel P.; Sarnaik, Ajit A.; Kochanek, Patrick M.; Clark, Robert S. B.

    2008-01-01

    Poly-ADP-ribosylation (PAR) of proteins by poly(ADP-ribose) polymerases (PARP) occurs after experimental traumatic brain injury (TBI) and modulates neurological outcome. Several promising pharmacological PARP inhibitors have been developed for use in humans, but there is currently no clinically relevant means of monitoring treatment effects. We therefore utilized an enzyme-linked immunosorbent assay (ELISA) to measure PAR-modified proteins in cerebrospinal fluid (CSF). CSF samples from 17 pediatric TBI and 15 control patients were plated overnight then incubated with polyclonal antibody against PAR. Histone-1, a PARP substrate, was incubated with active PARP, NAD, and nicked DNA, and served as the standard. Both peak and mean CSF PAR-modified protein were increased in TBI patients versus controls. Peak CSF PAR-modified protein levels occurred on day 1 and levels remained increased on day 2 after TBI. Increases in peak CSF PAR-modified protein concentrations were independently associated with age and male sex, but not initial Glasgow coma scale score, Glasgow outcome score, or mechanism of injury. The increase in PAR-modified proteins in CSF after TBI may be due to increased PARP activation, decreased PAR degradation, or both. Since PAR-modified protein concentration correlated with age and male sex, developmental and sex-dependent roles for PARP after TBI are implicated. PMID:18506195

  18. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    PubMed Central

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  19. Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin.

    PubMed

    Theerasilp, S; Hitotsuya, H; Nakajo, S; Nakaya, K; Nakamura, Y; Kurihara, Y

    1989-04-25

    The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536-11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor. PMID:2708331

  20. Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis.

    PubMed

    Han, Wenjuan; Yamauchi, Mika; Hasegawa, Urara; Noda, Masanori; Fukui, Kiichi; van der Vlies, André J; Uchiyama, Susumu; Uyama, Hiroshi

    2015-05-01

    Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column.

  1. The ctf13-30/CTF13 Genomic Haploinsufficiency Modifier Screen Identifies the Yeast Chromatin Remodeling Complex RSC, Which Is Required for the Establishment of Sister Chromatid Cohesion

    PubMed Central

    Baetz, Kristin K.; Krogan, Nevan J.; Emili, Andrew; Greenblatt, Jack; Hieter, Philip

    2004-01-01

    The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity. PMID:14729968

  2. Solution behavior of synthetic silk peptides and modified recombinant silk proteins

    NASA Astrophysics Data System (ADS)

    Foo, C. Wong Po; Bini, E.; Huang, J.; Lee, S. Y.; Kaplan, D. L.

    2006-02-01

    Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline β-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the α-helical structure and decreases in the β-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in β-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties.

  3. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

  4. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    PubMed Central

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-01-01

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model. PMID:27763556

  5. Free energy landscapes of encounter complexes in protein-protein association.

    PubMed Central

    Camacho, C J; Weng, Z; Vajda, S; DeLisi, C

    1999-01-01

    We report the computer generation of a high-density map of the thermodynamic properties of the diffusion-accessible encounter conformations of four receptor-ligand protein pairs, and use it to study the electrostatic and desolvation components of the free energy of association. Encounter complex conformations are generated by sampling the translational/rotational space of the ligand around the receptor, both at 5-A and zero surface-to-surface separations. We find that partial desolvation is always an important effect, and it becomes dominant for complexes in which one of the reactants is neutral or weakly charged. The interaction provides a slowly varying attractive force over a small but significant region of the molecular surface. In complexes with no strong charge complementarity this region surrounds the binding site, and the orientation of the ligand in the encounter conformation with the lowest desolvation free energy is similar to the one observed in the fully formed complex. Complexes with strong opposite charges exhibit two types of behavior. In the first group, represented by barnase/barstar, electrostatics exerts strong orientational steering toward the binding site, and desolvation provides some added adhesion within the local region of low electrostatic energy. In the second group, represented by the complex of kallikrein and pancreatic trypsin inhibitor, the overall stability results from the rather nonspecific electrostatic attraction, whereas the affinity toward the binding region is determined by desolvation interactions. PMID:10049302

  6. Endothelial Restoration of Receptor Activity-Modifying Protein 2 Is Sufficient to Rescue Lethality, but Survivors Develop Dilated Cardiomyopathy.

    PubMed

    Kechele, Daniel O; Dunworth, William P; Trincot, Claire E; Wetzel-Strong, Sarah E; Li, Manyu; Ma, Hong; Liu, Jiandong; Caron, Kathleen M

    2016-09-01

    RAMPs (receptor activity-modifying proteins) serve as oligomeric modulators for numerous G-protein-coupled receptors, yet elucidating the physiological relevance of these interactions remains complex. Ramp2 null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor signaling. We aimed to genetically rescue the Ramp2(-/-) lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with family B G-protein-coupled receptors in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2(-/-) mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2(-/-) Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking calcitonin receptor-like receptor. Yet, the hearts of Ramp2(-/-) Tg animals displayed dysregulation of family B G-protein-coupled receptors, including parathyroid hormone and glucagon receptors, as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional G-protein-coupled receptor pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/calcitonin receptor-like receptor signaling, in which future studies could elucidate novel and pharmacologically tractable pathways for treating cardiovascular diseases. PMID:27402918

  7. The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

    PubMed Central

    Cyster, J G; Shotton, D M; Williams, A F

    1991-01-01

    Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions. Images PMID:1706994

  8. RNA and protein complexes of trp RNA-binding attenuation protein characterized by mass spectrometry.

    PubMed

    Akashi, Satoko; Watanabe, Masahiro; Heddle, Jonathan G; Unzai, Satoru; Park, Sam-Yong; Tame, Jeremy R H

    2009-03-15

    We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution. PMID:19219981

  9. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  10. Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.

    PubMed

    Fisher, Robert J; Fivash, Matthew J; Stephen, Andrew G; Hagan, Nathan A; Shenoy, Shilpa R; Medaglia, Maxine V; Smith, Lindsey R; Worthy, Karen M; Simpson, John T; Shoemaker, Robert; McNitt, Karen L; Johnson, Donald G; Hixson, Catherine V; Gorelick, Robert J; Fabris, Daniele; Henderson, Louis E; Rein, Alan

    2006-01-01

    The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.

  11. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  12. Identification of human protein complexes from local sub-graphs of protein-protein interaction network based on random forest with topological structure features.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Zhou, Xuan; Dai, Zong; Zou, Xiao-Yong

    2012-03-01

    In the post-genomic era, one of the most important and challenging tasks is to identify protein complexes and further elucidate its molecular mechanisms in specific biological processes. Previous computational approaches usually identify protein complexes from protein interaction network based on dense sub-graphs and incomplete priori information. Additionally, the computational approaches have little concern about the biological properties of proteins and there is no a common evaluation metric to evaluate the performance. So, it is necessary to construct novel method for identifying protein complexes and elucidating the function of protein complexes. In this study, a novel approach is proposed to identify protein complexes using random forest and topological structure. Each protein complex is represented by a graph of interactions, where descriptor of the protein primary structure is used to characterize biological properties of protein and vertex is weighted by the descriptor. The topological structure features are developed and used to characterize protein complexes. Random forest algorithm is utilized to build prediction model and identify protein complexes from local sub-graphs instead of dense sub-graphs. As a demonstration, the proposed approach is applied to protein interaction data in human, and the satisfied results are obtained with accuracy of 80.24%, sensitivity of 81.94%, specificity of 80.07%, and Matthew's correlation coefficient of 0.4087 in 10-fold cross-validation test. Some new protein complexes are identified, and analysis based on Gene Ontology shows that the complexes are likely to be true complexes and play important roles in the pathogenesis of some diseases. PCI-RFTS, a corresponding executable program for protein complexes identification, can be acquired freely on request from the authors.

  13. Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives.

    PubMed

    Wen, Yuting; Guo, Zhenhuan; Du, Zhuo; Fang, Rong; Wu, Hongmei; Zeng, Xin; Wang, Chi; Feng, Min; Pan, Shirong

    2012-11-01

    Aiming to aid polyamidoamine (PAMAM, generation 4, PG4) to overcome gene delivery barriers like extrinsic serum inhibition, intrinsic cytotoxicity and lysosome digestion, histidine motifs modified PAMAM was prepared. The histidine activated PAMAM generation 4 (HPG4) was synthesized via aminolysis reaction and characterized by 1H NMR spectrum and MALDI-TOF-MS. Cytotoxicity profiles of HPG4 on MD-MB-231 cells were significantly improved in the form of polymer and polymer/DNA complexes comparing to PG4. The luciferase protein expression level of HPG4 was 20-, 2.7- and 1.2- fold higher than that of PG4, SuperFect and PEI 25k. Most importantly, flow cytometry and gene transfection studies showed that histidine motifs of HPG4 not only acted as enhancer for faster cellular uptake, but also played an important role on enhancing serum tolerance of the system on cellular uptake and transfection. Among the serum concentrations of 10%-50%, HPG4 showed 10-100 folds higher transfection efficiency than PG4. Intracellular fate observation conducted by confocal microscope provided visual and quantitative evidence that endsomal escape efficiency of HPG4 system was higher than that of PG4. Lastly, the endosomal escape mechanism of HPG4 system was analyzed by endosome destabilization and proton pump inhibition treatment. Collectively, compared to PG4/pDNA, HPG4/pDNA showed improvement on cellular uptake, serum tolerance, cytotoxicity profile, and endosomal escape.

  14. Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

    PubMed

    Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning

    2012-08-01

    Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

  15. Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)*

    PubMed Central

    Weston, Cathryn; Lu, Jing; Li, Naichang; Barkan, Kerry; Richards, Gareth O.; Roberts, David J.; Skerry, Timothy M.; Poyner, David; Pardamwar, Meenakshi; Reynolds, Christopher A.; Dowell, Simon J.; Willars, Gary B.; Ladds, Graham

    2015-01-01

    The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development. PMID:26198634

  16. Protein Adsorption on Chemically Modified Block Copolymer Nanodomains: Influence of Charge and Flow.

    PubMed

    Silverstein, Joshua S; Casey, Brendan J; Kofinas, Peter; Dair, Benita J

    2016-02-01

    Understanding the interactions of biomacromolecules with nanoengineered surfaces is vital for assessing material biocompatibility. This study focuses on the dynamics of protein adsorption on nanopatterned block copolymers (BCPs). Poly(styrene)-block-poly(1,2-butadiene) BCPs functionalized with an acid, amine, amide, or captopril moieties were processed to produce nanopatterned films. These films were characterized using water contact angle measurements and atomic force microscopy in air and liquid to determine how the modification process affected. wettability and swelling. Protein adsorption experiments were conducted under static and dynamic conditions via a quartz crystal microbalance with dissipation. Proteins of various size, charge, and stability were investigated to determine whether their physical characteristics affected adsorption. Significantly decreased contact angles were caused by selective swelling of modified BCP domains. The results indicate that nanopatterned chemistry and experimental conditions strongly impact adsorption dynamics. Depending on the structural stability of the protein, polyelectrolyte surfaces significantly increased adsorption over controls. Further analysis suggested that protein stability may correlate with dissipation versus frequency plots. PMID:27433605

  17. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins

    PubMed Central

    Song, Jing; Durrin, Linda K.; Wilkinson, Thomas A.; Krontiris, Theodore G.; Chen, Yuan

    2004-01-01

    Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2/Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V/I-X-V/I-V/I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2/Nup358 is responsible for the interaction between RanBP2/Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions. PMID:15388847

  18. Convergence Properties of Posttranslationally Modified Protein-Protein Switching Networks with Fast Decay Rates.

    PubMed

    Fan, Gaoyang; Cummins, Bree; Gedeon, Tomáš

    2016-06-01

    A significant conceptual difficulty in the use of switching systems to model regulatory networks is the presence of so-called "black walls," co-dimension 1 regions of phase space with a vector field pointing inward on both sides of the hyperplane. Black walls result from the existence of direct negative self-regulation in the system. One biologically inspired way of removing black walls is the introduction of intermediate variables that mediate the negative self-regulation. In this paper, we study such a perturbation. We replace a switching system with a higher-dimensional switching system with rapidly decaying intermediate proteins, and compare the dynamics between the two systems. We find that the while the individual solutions of the original system can be approximated for a finite time by solutions of a sufficiently close perturbed system, there are always solutions that are not well approximated for any fixed perturbation. We also study a particular example, where global basins of attraction of the perturbed system have a strikingly different form than those of the original system. We perform this analysis using techniques that are adapted to dealing with non-smooth systems. PMID:27271120

  19. 125I-labeled peptide mapping of some heat-modifiable proteins of the gonococcal outer membrane.

    PubMed Central

    Swanson, J

    1980-01-01

    Gonococci from opaque colonies have cell wall outer membrane proteins that are lacking from organisms which form transparent colonies. These "colony opacity-associated" proteins are among a group of "minor" proteins that exhibit heat modification of their apparent subunit molecular sizes, are easily extracted by deoxycholate, have apparent subunit molecular weights varying from 24,000 to 29,000 and are exposed on the surfaces of gonococci. Other minor proteins found on gonococci are the "leukocyte association proteins," whose presence correlates with reactivities of gonococci with human neutrophils. Several of the colony opacity-associated proteins and leukocyte association proteins were subjected to 125I-peptide mapping of protein bands separated by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. The structural similarities and differences among these heat-modifiable surface proteins were studied, as well as their similarities with the major protein of the gonococcal outer membrane. A relatively high apparent degree of structural homology is found among the heat-modifiable proteins from different strains of opaque colony gonococcal forms. There is also some apparent structural homology for 125I-peptides of heat-modifiable versus major proteins of the gonococcal outer membrane. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:6769820

  20. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    PubMed Central

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  1. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes.

    PubMed

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  2. Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

    PubMed Central

    Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

    2014-01-01

    The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes. PMID:25551646

  3. MOEPGA: A novel method to detect protein complexes in yeast protein-protein interaction networks based on MultiObjective Evolutionary Programming Genetic Algorithm.

    PubMed

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Song, Dan

    2015-10-01

    The identification of protein complexes in protein-protein interaction (PPI) networks has greatly advanced our understanding of biological organisms. Existing computational methods to detect protein complexes are usually based on specific network topological properties of PPI networks. However, due to the inherent complexity of the network structures, the identification of protein complexes may not be fully addressed by using single network topological property. In this study, we propose a novel MultiObjective Evolutionary Programming Genetic Algorithm (MOEPGA) which integrates multiple network topological features to detect biologically meaningful protein complexes. Our approach first systematically analyzes the multiobjective problem in terms of identifying protein complexes from PPI networks, and then constructs the objective function of the iterative algorithm based on three common topological properties of protein complexes from the benchmark dataset, finally we describe our algorithm, which mainly consists of three steps, population initialization, subgraph mutation and subgraph selection operation. To show the utility of our method, we compared MOEPGA with several state-of-the-art algorithms on two yeast PPI datasets. The experiment results demonstrate that the proposed method can not only find more protein complexes but also achieve higher accuracy in terms of fscore. Moreover, our approach can cover a certain number of proteins in the input PPI network in terms of the normalized clustering score. Taken together, our method can serve as a powerful framework to detect protein complexes in yeast PPI networks, thereby facilitating the identification of the underlying biological functions.

  4. Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

    PubMed Central

    Holt, Matthew; Muir, Tom

    2016-01-01

    Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments. PMID:25784050

  5. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes

    PubMed Central

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly. PMID:26868667

  6. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes.

    PubMed

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly.

  7. Carboxymethylchitosan covalently modified capillary column for open tubular capillary electrochromatography of basic proteins and opium alkaloids.

    PubMed

    Zhou, Sunying; Tan, Jingjing; Chen, Qinhua; Lin, Xucong; Lü, Haixia; Xie, Zenghong

    2010-12-24

    A novel open tubular (OT) column covalently modified with hydrophilic polysaccharide, carboxymethylchitosan (CMC) as stationary phase has been developed, and employed for the separations of basic proteins and opium alkaloids by capillary electrochromatography (CEC). With the procedures including the silanization of 3-aminopropyltrimethoxysilane (APTS) and the combination of glutaraldehyde with amino-silylated silica surface and CMC, CMC was covalently bonded on the capillary inner wall and exhibited a remarkable tolerance and chemical stability against 0.1 mol/L HCl, 0.1 mol/L NaOH or some organic solvents. By varying the pH values of running buffer, a cathodic or anodic EOF could be gained in CMC modified column. With anodic EOF mode (pH<4.3), favorable separations of basic proteins (trypsin, ribonuclease A, lysozyme and cytochrome C) were successfully achieved with high column efficiencies ranging from 97,000 to 182,000 plates/m, and the undesired adsorptions of basic proteins on the inter-wall of capillary could be avoided. Good repeatability was gained with RSD of the migration time less than 1.3% for run-to-run (n=5) and less than 3.2% for day-to-day (n=3), RSD of peak area was less than 5.6% for run-to-run (n=5) and less than 8.8% for day-to-day (n=3). With cathodic EOF mode (pH>4.3), four opium alkaloids were also baseline separated in phosphate buffer (50 mmol/L, pH 6.0) with column efficiencies ranging from 92,000 to 132,000 plates/m. CMC-bonded OT capillary column might be used as an alternative medium for the further analysis of basic proteins and alkaline analytes.

  8. Ion-ion reactions with fixed-charge modified proteins to produce ions in a single, very high charge state

    NASA Astrophysics Data System (ADS)

    Frey, Brian L.; Krusemark, Casey J.; Ledvina, Aaron R.; Coon, Joshua J.; Belshaw, Peter J.; Smith, Lloyd M.

    2008-10-01

    Electrospray ionization (ESI) of denatured proteins produces a mass spectrum with a broad distribution of multiply charged ions. Attaching fixed positive charges, specifically quaternary ammonium groups, to proteins at their carboxylic acid groups generates substantially higher charge states compared to the corresponding unmodified proteins in positive-mode ESI. Ion-ion reactions of these modified proteins with reagent anions leads to charge reduction by proton transfer. These proton transfer reactions cannot remove charge from the quaternary ammonium groups, which do not have a proton to transfer to the anion. Thus, one might expect charge reduction to stop at a single charge state equal to the number of fixed charges on the modified protein. However, ion-ion reactions yield charge states lower than this number of fixed charges due to anion attachment (adduction) to the proteins. Charge reduction via ion-molecule reactions involving gas-phase bases also give adducts on the modified protein ions in low charge states. Such adducts are avoided by keeping the ions in charge states well above the number of fixed charges. In the present work protein ions were selectively "parked" within an ion trap mass spectrometer in a high charge state by mild radiofrequency excitation that dramatically slows their ion-ion reaction rate--a technique termed "ion parking". The combination of ion parking with the fixed-charge modified proteins permits generation of a large population of ions in a single, very high charge state.

  9. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  10. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  11. A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing*

    PubMed Central

    Cordeiro, Tiago N.; García, Jesús; Bernadó, Pau; Millet, Oscar; Pons, Miquel

    2015-01-01

    The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related. PMID:26085102

  12. EXPERIMENT-GUIDED MOLECULAR MODELING OF PROTEIN-PROTEIN COMPLEXES INVOLVING GPCRS

    PubMed Central

    Kufareva, Irina; Handel, Tracy M.

    2015-01-01

    Summary Experimental structure determination for G protein coupled receptors (GPCRs) and especially their complexes with protein and peptide ligands is at its infancy. In the absence of complex structures, molecular modeling and docking play a large role not only by providing a proper 3D context for interpretation of biochemical and biophysical data, but also by prospectively guiding experiments. Experimentally confirmed restraints may help improve the accuracy and information content of the computational models. Here we present a hybrid molecular modeling protocol that integrates heterogeneous experimental data with force field-based calculations in the stochastic global optimization of the conformations and relative orientations of binding partners. Some experimental data, such as pharmacophore-like chemical fields or disulfide-trapping restraints, can be seamlessly incorporated in the protocol, while other types of data are more useful at the stage of solution filtering. The protocol was successfully applied to modeling and design of a stable construct that resulted in crystallization of the first complex between a chemokine and its receptor. Examples from this work are used to illustrate the steps of the protocol. The utility of different types of experimental data for modeling and docking is discussed and caveats associated with data misinterpretation are highlighted. PMID:26260608

  13. Identifying functions of protein complexes based on topology similarity with random forest.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2014-03-01

    Elucidating the functions of protein complexes is critical for understanding disease mechanisms, diagnosis and therapy. In this study, based on the concept that protein complexes with similar topology may have similar functions, we firstly model protein complexes as weighted graphs with nodes representing the proteins and edges indicating interaction between proteins. Secondly, we use topology features derived from the graphs to characterize protein complexes based on the graph theory. Finally, we construct a predictor by using random forest and topology features to identify the functions of protein complexes. Effectiveness of the current method is evaluated by identifying the functions of mammalian protein complexes. And then the predictor is also utilized to identify the functions of protein complexes retrieved from human protein-protein interaction networks. We identify some protein complexes with significant roles in the occurrence of tumors, vesicles and retinoblastoma. It is anticipated that the current research has an important impact on pathogenesis and the pharmaceutical industry. The source code of Matlab and the dataset are freely available on request from the authors. PMID:24389559

  14. [Castaneda-de Leval's modified Fontan operation in complex cyanotic defects with subsequent percutaneous transluminal embolization of systemic pulmonary collaterals].

    PubMed

    Schubel, B; Gliech, V; Warnke, H; Göldner, B

    1990-01-01

    The case of a five year old boy with a double-inlet-left-ventricle and the aorta in 1-malposition, large VSD, ASD and pulmonary stenosis is reported, that showed after modified Fontan surgery severe effusions and a severe protein loss syndrome. Aortopulmonary collaterals were detected as the cause of this and successfully treated in several sessions by percutaneous transluminal embolization.

  15. Complex-scaling of screened Coulomb potentials for resonance calculations utilizing the modified Bessel functions

    NASA Astrophysics Data System (ADS)

    Jiao, Li-Guang; Ho, Yew Kam

    2014-05-01

    The screened Coulomb potential (SCP) has been extensively used in atomic physics, nuclear physics, quantum chemistry and plasma physics. However, an accurate calculation for atomic resonances under SCP is still a challenging task for various methods. Within the complex-scaling computational scheme, we have developed a method utilizing the modified Bessel functions to calculate doubly-excited resonances in two-electron atomic systems with configuration interaction-type basis. To test the validity of our method, we have calculated S- and P-wave resonance states of the helium atom with various screening strengths, and have found good agreement with earlier calculations using different methods. Our present method can be applied to calculate high-lying resonances associated with high excitation thresholds of the He+ ion, and with high-angular-momentum states. The derivation and calculation details of our present investigation together with new results of high-angular-momentum states will be presented at the meeting. Supported by NSC of Taiwan.

  16. Microencapsulation of stearidonic acid soybean oil in Maillard reaction-modified complex coacervates.

    PubMed

    Ifeduba, Ebenezer A; Akoh, Casimir C

    2016-05-15

    The antioxidant capacity of Maillard reaction (MR)-modified gelatin (GE)-gum arabic (GA) coacervates was optimized to produce microcapsules with superior oxidative stability compared to the unmodified control. MR was used to crosslink GE and GA, with or without maltodextrin (MD), to produce anti-oxidative Maillard reaction products (MRP) which was used to encapsulate stearidonic acid soybean oil (SDASO) by complex coacervation. Biopolymer blends (GE-GA [1:1, w/w] or GE-GA-MD [2:2:1, w/w/w]) were crosslinked by dry-heating at 80°C for 4, 8, or 16h. Relationships between the extent of browning, Trolox equivalent antioxidant capacity (TEAC), and the total oxidation (TOTOX) of encapsulated SDASO were fitted to quadratic models. The [GE-GA-MD] blends exhibited higher browning rates and TEAC values than corresponding [GE-GA] blends. Depending on the type of biopolymer blend and dry-heating time, TOTOX values of SDASO in MRP-derived microcapsules were 29-87% lower than that of the non-crosslinked control after 30 days of storage.

  17. Thiazole/oxazole-modified microcins: complex natural products from ribosomal templates

    PubMed Central

    Melby, Joel O.; Nard, Nathan J.; Mitchell, Douglas A.

    2014-01-01

    With billions of years of evolution under its belt, Nature has been expanding and optimizing its biosynthetic capabilities. Chemically complex secondary metabolites continue to challenge and inspire today’s most talented synthetic chemists. A brief glance at these natural products, especially the substantial structural variation within a class of compounds, clearly demonstrates that Nature has long played the role of medicinal chemist. The recent explosion in genome sequencing has expanded our appreciation of natural product space and the vastness of uncharted territory that remains. One small corner of natural product chemical space is occupied by the recently dubbed thiazole/oxazole-modified microcins (TOMMs), which are ribosomally produced peptides with posttranslationally installed heterocycles derived from cysteine, serine and threonine residues. As with other classes of natural products, the genetic capacity to synthesize TOMMs has been widely disseminated among bacteria. Over the evolutionary timescale, Nature has tested countless random mutations and selected for gain of function in TOMM biosynthetic gene clusters, yielding several privileged molecular scaffolds. Today, this burgeoning class of natural products encompasses a structurally and functionally diverse set of molecules (i.e. microcin B17, cyanobactins, and thiopeptides). TOMMs presumably provide their producers with an ecological advantage. This advantage can include chemical weapons wielded in the battle for nutrients, disease-promoting virulence factors, or compounds presumably beneficial for symbiosis. Despite this plethora of functions, many TOMMs await experimental interrogation. This review will focus on the biosynthesis and natural combinatorial diversity of the TOMM family. PMID:21429787

  18. Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

    PubMed

    Miodek, A; Castillo, G; Hianik, T; Korri-Youssoufi, H

    2014-06-15

    This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%. PMID:24480126

  19. Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

    PubMed

    Miodek, A; Castillo, G; Hianik, T; Korri-Youssoufi, H

    2014-06-15

    This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

  20. Amperometric immunosensor based on a protein A/deposited gold nanocrystals modified electrode for carbofuran detection.

    PubMed

    Sun, Xia; Zhu, Ying; Wang, Xiangyou

    2011-01-01

    In this paper, an amperometric immunosensor modified with protein A/deposited gold nanocrystals (DpAu) was developed for the ultrasensitive detection of carbofuran residues. First, DpAu were electrodeposited onto the Au electrode surface to absorb protein A (PA) and improve the electrode conductivity. Then PA was dropped onto the surface of DpAu film, used for binding antibody Fc fragments. Next, anti-carbofuran monoclonal antibody was immobilized on the PA modified electrode. Finally, bovine serum albumin (BSA) was employed to block the possible remaining active sites avoiding any nonspecific adsorption. The fabrication procedure of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. With the excellent electroconductivity of DpAu and the PA's oriented immobilization of antibodies, a highly efficient immuno-reaction and detection sensitivity could be achieved. The influences of the electrodeposition time of DpAu, pH of the detection solution and incubation time on the current response of the fabricated immunosensor were investigated. Under optimized conditions, the current response was proportional to the concentration of carbofuran which ranged from 1 to 100 ng/mL and 100 ng/mL to 100 μg/mL. The detection limit was 0.1924 ng/mL. The proposed carbofuran immnuosensor exhibited high specificity, reproducibility, stability and regeneration performance, which may open a new door for ultrasensitive detection of carbofuran residues in vegetables and fruits.

  1. Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

    PubMed

    Di Sansebastiano, Gian Pietro; Rizzello, Francesca; Durante, Miriana; Caretto, Sofia; Nisi, Rossella; De Paolis, Angelo; Faraco, Marianna; Montefusco, Anna; Piro, Gabriella; Mita, Giovanni

    2015-05-20

    Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

  2. Variants within the SP110 nuclear body protein modify risk of canine degenerative myelopathy

    PubMed Central

    Ivansson, Emma L.; Kozyrev, Sergey V.; Murén, Eva; Körberg, Izabella Baranowska; Swofford, Ross; Koltookian, Michele; Tonomura, Noriko; Zeng, Rong; Kolicheski, Ana L.; Hansen, Liz; Katz, Martin L.; Johnson, Gayle C.; Johnson, Gary S.; Coates, Joan R.; Lindblad-Toh, Kerstin

    2016-01-01

    Canine degenerative myelopathy (DM) is a naturally occurring neurodegenerative disease with similarities to some forms of amyotrophic lateral sclerosis (ALS). Most dogs that develop DM are homozygous for a common superoxide dismutase 1 gene (SOD1) mutation. However, not all dogs homozygous for this mutation develop disease. We performed a genome-wide association analysis in the Pembroke Welsh Corgi (PWC) breed comparing DM-affected and -unaffected dogs homozygous for the SOD1 mutation. The analysis revealed a modifier locus on canine chromosome 25. A haplotype within the SP110 nuclear body protein (SP110) was present in 40% of affected compared with 4% of unaffected dogs (P = 1.5 × 10−5), and was associated with increased probability of developing DM (P = 4.8 × 10−6) and earlier onset of disease (P = 1.7 × 10−5). SP110 is a nuclear body protein involved in the regulation of gene transcription. Our findings suggest that variations in SP110-mediated gene transcription may underlie, at least in part, the variability in risk for developing DM among PWCs that are homozygous for the disease-related SOD1 mutation. Further studies are warranted to clarify the effect of this modifier across dog breeds. PMID:27185954

  3. Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids

    PubMed Central

    Tookmanian, Elise M.; Fenlon, Edward E.; Brewer, Scott H.

    2015-01-01

    Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein. PMID:26478813

  4. Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography.

    PubMed

    Zhang, Yamin; Wang, Wentao; Ma, Xiangdong; Jia, Li

    2016-11-01

    Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO2) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO2 coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti(4+) to boost the nucleation and growth of TiO2 film. The as-prepared TiO2 coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO2 coating was investigated. The TiO2 coated OT column was used for successful separation of two variants of β-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white. PMID:27555440

  5. Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries.

    PubMed

    Karim, M R; Shinagawa, S; Takagi, T

    1994-01-01

    Electrophoretic mobilities of various proteins or peptides complexed with sodium dodecyl sulfate (SDS) were determined by free solution capillary electrophoresis. The complexes formed between SDS and protein polypeptide showed electrophoretic mobilities virtually insensitive to the protein molecular weight. On the other hand, scattered and larger negative electrophoretic mobilities were observed for peptides of molecular weights less than 10000. Mobility differences as small as 0.3-3% among the three differently modified derivatives of bovine serum albumin could also be successfully determined due to the high resolving power of capillary electrophoresis.

  6. Surface forces and protein adsorption on dextran- and polyethylene glycol-modified polydimethylsiloxane.

    PubMed

    Farrell, Megan; Beaudoin, Stephen

    2010-12-01

    Dextran and polyethylene glycol (PEG) are often covalently bound to the surface of polydimethylsiloxane (PDMS) for the purpose of modifying its hydrophilicity and biocompatibility. In this work, the effects of the dextran and PEG on the morphology, wetting, and surface charge of the resulting surfaces were quantified and correlated with changes in the amount of fibrinogen and albumin adsorbed from aqueous solution. PDMS films were functionalized in a microwave oxygen plasma to create surface hydroxyl groups that were subsequently aminated by incubation in a (3-aminopropyl)trimethoxysilane (APTES) solution. Oxidized dextran and PEG-aldehyde were linked to the surface amines via reductive amination. This process resulted in low surface coverage of immobilized PEG in the end-on conformation and a more uniform and dense distribution of side-on immobilized dextran. The immobilized dextran reduced the contact angle of the PDMS film from 109° to 80° and neutralized the zeta potential over the pH range from 3 to 11. An atomic force microscope was used to measure the interaction force between the modified PDMS and a model hydrophobic surface (polystyrene latex) and a model hydrophilic surface (silica) in aqueous solution to show that van der Waals and hydrophobic attractive forces are the dominant forces for protein adsorption in this system. The PEG- and dextran-modified PDMS were exposed to BSA and fibrinogen to test their resistance to protein adsorption. The coatings were ineffective at reducing the adsorption of either molecule, and the dextran-modification of the PDMS caused more BSA to adsorb than in the case of the unmodified PDMS. PMID:20801620

  7. Short transmembrane domains with high-volume exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex.

    PubMed

    Quiroga, Rodrigo; Trenchi, Alejandra; González Montoro, Ayelén; Valdez Taubas, Javier; Maccioni, Hugo J F

    2013-12-01

    It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the Golgi SNARE protein Sft1 and the plasma membrane SNARE protein Sso1 from Saccharomyces cerevisiae as model proteins, we modified the length of their TMDs and the volume of their exoplasmic hemi-TMD, and determined their subcellular localization both in yeast and mammalian cells. We found that short TMDs with high-volume exoplasmic hemi-TMDs confer Golgi membrane residence, whereas TMDs with low-volume exoplasmic hemi-TMDs, either short or long, confer plasma membrane residence to these proteins. Results indicate that the shape of the exoplasmic hemi-TMD, in addition to the length of the entire TMD, determine retention in the Golgi or exit to the plasma membrane of Type II membrane proteins.

  8. Ion mobility mass spectrometry of proteins in a modified commercial mass spectrometer

    NASA Astrophysics Data System (ADS)

    Thalassinos, K.; Slade, S. E.; Jennings, K. R.; Scrivens, J. H.; Giles, K.; Wildgoose, J.; Hoyes, J.; Bateman, R. H.; Bowers, M. T.

    2004-08-01

    Ion mobility has emerged as an important technique for determining biopolymer conformations in solvent free environments. These experiments have been nearly exclusively performed on home built systems. In this paper we describe modifications to a commercial high performance mass spectrometer, the Waters UK "Ultima" Q-Tof, that allows high sensitivity measurement of peptide and protein cross sections. Arrival time distributions are obtained for a series of peptides (bradykinin, LHRH, substance P, bombesin) and proteins (bovine and equine cytochrome c, myoglobin, [alpha]-lactalbumin) with good agreement found with literature cross sections where available. In complex ATD's, mass spectra can be obtained for each feature confirming assignments. The increased sensitivity of the commercial instrument is retained along with the convenience of the data system, crucial features for analysis of protein misfolding systems.

  9. Piezo dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein interactions.

    PubMed

    Klenkar, Goran; Valiokas, Ramûnas; Lundström, Ingemar; Tinazli, Ali; Tampé, Robert; Piehler, Jacob; Liedberg, Bo

    2006-06-01

    The fabrication of a novel biochip, designed for dissection of multiprotein complex formation, is reported. An array of metal chelators has been produced by piezo dispensing of a bis-nitrilotriacetic acid (bis-NTA) thiol on evaporated gold thin films, prestructured with a microcontact printed grid of eicosanethiols. The bis-NTA thiol is mixed in various proportions with an inert, tri(ethylene glycol) hexadecane thiol, and the thickness and morphological homogeneity of the dispensed layers are characterized by imaging ellipsometry before and after back-filling with the same inert thiol and subsequent rinsing. It is found that the dispensed areas display a monotonic increase in thickness with increasing molar fraction of bis-NTA in the dispensing solution, and they are consistently a few Angströms thicker than those prepared at the same molar fraction by solution self-assembly under equilibrium-like conditions. The bulkiness of the bis-NTA tail group and the short period of time available for chemisorption and in-plane organization of the dispensed thiols are most likely responsible for the observed difference in thickness. Moreover, the functional properties of this biochip are demonstrated by studying multiple protein-protein interactions using imaging surface plasmon resonance. The subunits of the type I interferon receptor are immobilized as a composition array determined by the surface concentration of bis-NTA in the array elements. Ligand dissociation kinetics depends on the receptor surface concentration, which is ascribed to the formation of a ternary complex by simultaneous interaction of the ligand with the two receptor subunits. Thus, multiplexed monitoring of binding phenomena at various compositions (receptor densities) offers a powerful tool to dissect protein-protein interactions.

  10. Organic Chemistry Applied to Synthetic Proteins: Modifying the Vicinity of the Isopeptide Bond Revealed Differential Behavior of Ubiquitin Chains with Interacting Proteins

    PubMed Central

    Haj-Yahya, Najat; Haj-Yahya, Mahmood; Castañeda, Carlos A.; Spasser, Liat; Hemantha, Hosahalli P.; Jbara, Muhammad; Penner, Marlin; Ciechanover, Aaron; Fushman, David

    2013-01-01

    In Every Direction Chemical synthesis of proteins allowed the synthesis of ubiquitin chains modified in the vicinity of the isopeptide peptide to examine their behavior with deubiquitinases and ubiquitin binding domains. Our results set the ground for the generation of unique probes for studying the interactions of these chains with various ubiquitin-interacting proteins. PMID:24006204

  11. Human-Modified Permafrost Complexes in Urbanized Areas of the Russian North

    NASA Astrophysics Data System (ADS)

    Grebenets, V. I.; Streletskiy, D. A.

    2013-12-01

    Economic development in permafrost regions is accompanied by modification of natural geocryological conditions. Drastic landscape transformations in urbanized areas on permafrost are characterized by changes of heat and moisture exchange in permafrost - atmosphere system, and by engineering and technogenic influence upon the frozen ground, leading to alteration of its physical, thermal and mechanical properties. In northern cities this leads to overall increase of ground temperature relative to undisturbed areas and intensification of hazardous cryogenic processes in areas under engineering development, which together leads to reduction in stability of geotechnical environment. For example, deformations of structures in Norilsk district, Northern Siberia, in the last 15 years, became much more abundant than those revealed throughout the previous 50 years. About 250 large buildings in the local towns were deformed considerably due to deterioration of geocryological conditions, about 100 structures were functioning in emergency state, and almost 50 nine- and five-storey houses, built in the 1960-80s, have been recently disassembled. Increase in accident risk for various facilities (water and oil pipelines, industrial enterprises, etc.) enhances the technogenic pressure on permafrost, leading to the new milestone of changes in permafrost characteristics, i.e. to creation of 'another reality' of geocryological conditions. Social and natural factors dictate clustered spatial pattern of industrial development in permafrost regions. Cryogenic processes within the urban areas on permafrost are seldom similar with those under the natural conditions as intensity, duration and extent of the processes changes under technogenic impacts. Moreover, new cryogenic processes and phenomena may occur, which have not been typical for a given region. This makes mapping and characterization of these processes difficult task. Peculiar natural-technogenic geocryological complexes (NTGC

  12. Folding analysis of the most complex Stevedore's protein knot.

    PubMed

    Wang, Iren; Chen, Szu-Yu; Hsu, Shang-Te Danny

    2016-01-01

    DehI is a homodimeric haloacid dehalogenase from Pseudomonas putida that contains the most complex 61 Stevedore's protein knot within its folding topology. To examine how DehI attains such an intricate knotted topology we combined far-UV circular dichroism (CD), intrinsic fluorescence spectroscopy and small angle X-ray scattering (SAXS) to investigate its folding mechanism. Equilibrium unfolding of DehI by chemical denaturation indicated the presence of two highly populated folding intermediates, I and I'. While the two intermediates vary in secondary structure contents and tertiary packing according to CD and intrinsic fluorescence, respectively, their overall dimension and compactness are similar according to SAXS. Three single-tryptophan variants (W34, W53, and W196) were generated to probe non-cooperative unfolding events localized around the three fluorophores. Kinetic fluorescence measurements indicated that the transition from the intermediate I' to the unfolded state is rate limiting. Our multiparametric folding analyses suggest that DehI unfolds through a linear folding pathway with two distinct folding intermediates by initial hydrophobic collapse followed by nucleation condensation, and that knotting precedes the formation of secondary structures. PMID:27527519

  13. Evidence for a vasopressin receptor-GTP binding protein complex

    SciTech Connect

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

  14. The Ogura sterility-inducing protein forms a large complex without interfering with the oxidative phosphorylation components in rapeseed mitochondria.

    PubMed

    Duroc, Yann; Hiard, Sophie; Vrielynck, Nathalie; Ragu, Sandrine; Budar, Françoise

    2009-05-01

    The Ogura cytoplasmic male sterility causing protein, ORF138, was found to be part of a complex with an apparent size of over 750 kDa in the inner membrane of mitochondria of sterile plants. ORF138 did not colocalize with any of the oxidative phosphorylation complexes, nor did its presence modify their apparent size or amount, compared to samples from fertile isogenic plants. We attempted to detect potential proteins or nucleic acids that could be involved in the large ORF138 complex by 2D PAGE, immunoprecipitation and nuclease treatments of native extracts. All our results suggest that the ORF138 protein is the main, if not only, component of this large complex. The capacities of complexes I, II, IV, and ATP synthase were identical in samples from sterile and fertile plants. Isolated mitochondria from sterile plants showed a higher oxygen consumption than those from fertile plants. In vivo respiration measurements suggest that the difference in O(2) consumption measured at the organelle level is compensated at the cell/tissue level, completely in leaves, but only partially in male reproductive organs.

  15. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes During In Vitro Pepsin Digestion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  16. Morphological remodeling of C. elegans neurons during aging is modified by compromised protein homeostasis

    PubMed Central

    Vayndorf, Elena M; Scerbak, Courtney; Hunter, Skyler; Neuswanger, Jason R; Toth, Marton; Parker, J Alex; Neri, Christian; Driscoll, Monica; Taylor, Barbara E

    2016-01-01

    Understanding cellular outcomes, such as neuronal remodeling, that are common to both healthy and diseased aging brains is essential to the development of successful brain aging strategies. Here, we used Caenorhabdits elegans to investigate how the expression of proteotoxic triggers, such as polyglutamine (polyQ)-expanded huntingtin and silencing of proteostasis regulators, such as the ubiquitin–proteasome system (UPS) and protein clearance components, may impact the morphological remodeling of individual neurons as animals age. We examined the effects of disrupted proteostasis on the integrity of neuronal cytoarchitecture by imaging a transgenic C. elegans strain in which touch receptor neurons express the first 57 amino acids of the human huntingtin (Htt) gene with expanded polyQs (128Q) and by using neuron-targeted RNA interference in adult wild-type neurons to knockdown genes encoding proteins involved in proteostasis. We found that proteostatic challenges conferred by polyQ-expanded Htt and knockdown of specific genes involved in protein homeostasis can lead to morphological changes that are restricted to specific domains of specific neurons. The age-associated branching of PLM neurons is suppressed by N-ter polyQ-expanded Htt expression, whereas ALM neurons with polyQ-expanded Htt accumulate extended outgrowths and other soma abnormalities. Furthermore, knockdown of genes important for ubiquitin-mediated degradation, lysosomal function, and autophagy modulated these age-related morphological changes in otherwise normal neurons. Our results show that the expression of misfolded proteins in neurodegenerative disease such as Huntington’s disease modifies the morphological remodeling that is normally associated with neuronal aging. Our results also show that morphological remodeling of healthy neurons during aging can be regulated by the UPS and other proteostasis pathways. Collectively, our data highlight a model in which morphological remodeling during

  17. Chemically modified diamond-like carbon (DLC) for protein enrichment and profiling by MALDI-MS.

    PubMed

    Najam-ul-Haq, M; Rainer, M; Huck, C W; Ashiq, M N; Bonn, G K

    2012-08-01

    The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.

  18. Cationic versus anionic surfactant in tuning the structure and interaction of nanoparticle, protein, and surfactant complexes.

    PubMed

    Mehan, Sumit; Aswal, Vinod K; Kohlbrecher, Joachim

    2014-08-26

    The structure and interaction in complexes of anionic Ludox HS40 silica nanoparticle, anionic bovine serum albumin (BSA) protein, and cationic dodecyl trimethylammonium bromide (DTAB) surfactant have been studied using small-angle neutron scattering (SANS). The results are compared with similar complexes having anionic sodium dodecyl sulfate (SDS) surfactant (Mehan, S; Chinchalikar, A. J.; Kumar, S.; Aswal, V. K.; Schweins, R. Langmuir 2013, 29, 11290). In both cases (DTAB and SDS), the structure in nanoparticle-protein-surfactant complexes is predominantly determined by the interactions of the individual two-component systems. The nanoparticle-surfactant (mediated through protein-surfactant complex) and protein-surfactant interactions for DTAB, but nanoparticle-protein (mediated through protein-surfactant complex) and protein-surfactant interactions for SDS, are found to be responsible for the resultant structure of nanoparticle-protein-surfactant complexes. Irrespective of the charge on the surfactant, the cooperative binding of surfactant with protein leads to micellelike clusters of surfactant formed along the unfolded protein chain. The adsorption of these protein-surfactant complexes for DTAB on oppositely charged nanoparticles gives rise to the protein-surfactant complex-mediated aggregation of nanoparticles (similar to that of DTAB surfactant). It is unlike that of depletion-induced aggregation of nanoparticles with nonadsorption of protein-surfactant complexes for SDS in similarly charged nanoparticle systems (similar to that of protein alone). The modifications in nanoparticle aggregation as well as unfolding of protein in these systems as compared to the corresponding two-component systems have also been examined by selectively contrast matching the constituents.

  19. 193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology.

    PubMed

    Morrison, Lindsay J; Brodbelt, Jennifer S

    2016-08-31

    Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins. PMID:27480400

  20. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity.

    PubMed

    Brehme, Marc; Voisine, Cindy

    2016-08-01

    Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  1. Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

    NASA Astrophysics Data System (ADS)

    Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

    2005-04-01

    This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

  2. A genetically modified protein-based hydrogel for 3D culture of AD293 cells.

    PubMed

    Du, Xiao; Wang, Jingyu; Diao, Wentao; Wang, Ling; Long, Jiafu; Zhou, Hao

    2014-01-01

    Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence 'GRGDSP' to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery. PMID:25233088

  3. Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin.

    PubMed

    Tian, Qing-Yun; Zhao, Yun-Peng; Liu, Chuan-ju

    2012-01-17

    Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.

  4. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    PubMed Central

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  5. Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation

    PubMed Central

    Batulan, Zarah; Pulakazhi Venu, Vivek Krishna; Li, Yumei; Koumbadinga, Geremy; Alvarez-Olmedo, Daiana Gisela; Shi, Chunhua; O’Brien, Edward R.

    2016-01-01

    Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis. PMID:27507972

  6. Overexpression of a modified amaranth protein in Escherichia coli with minimal media and lactose as inducer.

    PubMed

    Morales-Camacho, Jocksan Ismael; Dominguez-Dominguez, Jorge; Paredes-Lopez, Octavio

    2013-04-01

    In this research it was attempted to overexpress the acidic subunit, from the 11S amaranth seed globulin termed amarantin, modified with antihypertensive peptides in Escherichia coli Rosetta (DE3) by manipulating some factors in batch fermenter such as growth medium composition, inducer (isopropyl β-D-thiogalactopyranoside [IPTG] or lactose), air flow, cultivation temperature, agitation speed and induction time. The possibility of using several minimal media and lactose as inducer to increase yields of the recombinant protein was investigated. Previous fermentations at flask level showed that two minimal culture media (A6 and A7) and 0.5% (w/v) lactose presented high yields of the engineered protein expression. Thus, the latter two media were tested at fermenter level, the lactose inducer, and different environmental conditions. Factors with significant effects were identified by Plackett-Burman design with center points and were adjusted at the level suggested and the yields of the recombinant protein were increased from 303.2 to 1,531 mg L(-1) in A6 and from 363.4 to 1,681 mg L(-1) in A7. Unlike some patents where the highest productivity was achieved at 24 h or afterwards, in this research the best productivity of the recombinant acidic subunit was attained at 4 and 6 h of induction using both media, respectively. PMID:23294401

  7. Protein complex prediction via improved verification methods using constrained domain-domain matching.

    PubMed

    Zhao, Yang; Hayashida, Morihiro; Nacher, Jose C; Nagamochi, Hiroshi; Akutsu, Tatsuya

    2012-01-01

    Identification of protein complexes within protein-protein interaction networks is one of the important objectives in functional genomics. Ozawa et al. proposed a verification method of protein complexes by introducing a structural constraint. In this paper, we propose an improved integer programming-based method based on the idea that a candidate complex should not be divided into many small complexes, and combination methods with maximal components and extreme sets. The results of computational experiments suggest that our methods outperform the method by Ozawa et al. We prove that the verification problems are NP-hard, which justifies the use of integer programming. PMID:22961452

  8. Modified Low Density Lipoprotein and Lipoprotein-Containing Circulating Immune Complexes as Diagnostic and Prognostic Biomarkers of Atherosclerosis and Type 1 Diabetes Macrovascular Disease

    PubMed Central

    Orekhov, Alexander N.; Bobryshev, Yuri V.; Sobenin, Igor A.; Melnichenko, Alexandra A.; Chistiakov, Dimitry A.

    2014-01-01

    In atherosclerosis; blood low-density lipoproteins (LDL) are subjected to multiple enzymatic and non-enzymatic modifications that increase their atherogenicity and induce immunogenicity. Modified LDL are capable of inducing vascular inflammation through activation of innate immunity; thus, contributing to the progression of atherogenesis. The immunogenicity of modified LDL results in induction of self-antibodies specific to a certain type of modified LDL. The antibodies react with modified LDL forming circulating immune complexes. Circulating immune complexes exhibit prominent immunomodulatory properties that influence atherosclerotic inflammation. Compared to freely circulating modified LDL; modified LDL associated with the immune complexes have a more robust atherogenic and proinflammatory potential. Various lipid components of the immune complexes may serve not only as diagnostic but also as essential predictive markers of cardiovascular events in atherosclerosis. Accumulating evidence indicates that LDL-containing immune complexes can also serve as biomarker for macrovascular disease in type 1 diabetes. PMID:25050779

  9. Qualitative and Quantitative Protein Complex Prediction Through Proteome-Wide Simulations.

    PubMed

    Rizzetto, Simone; Priami, Corrado; Csikász-Nagy, Attila

    2015-10-01

    Despite recent progress in proteomics most protein complexes are still unknown. Identification of these complexes will help us understand cellular regulatory mechanisms and support development of new drugs. Therefore it is really important to establish detailed information about the composition and the abundance of protein complexes but existing algorithms can only give qualitative predictions. Herein, we propose a new approach based on stochastic simulations of protein complex formation that integrates multi-source data--such as protein abundances, domain-domain interactions and functional annotations--to predict alternative forms of protein complexes together with their abundances. This method, called SiComPre (Simulation based Complex Prediction), achieves better qualitative prediction of yeast and human protein complexes than existing methods and is the first to predict protein complex abundances. Furthermore, we show that SiComPre can be used to predict complexome changes upon drug treatment with the example of bortezomib. SiComPre is the first method to produce quantitative predictions on the abundance of molecular complexes while performing the best qualitative predictions. With new data on tissue specific protein complexes becoming available SiComPre will be able to predict qualitative and quantitative differences in the complexome in various tissue types and under various conditions.

  10. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

  11. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

    PubMed Central

    LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

    2015-01-01

    Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

  12. Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

    PubMed Central

    Wang, Jianlong; Cantor, Alan B.; Orkin, Stuart H.

    2009-01-01

    In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. PMID:19306258

  13. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability.

    PubMed

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-14

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ(-/-) mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper-tyrosine-phosphorylated in the PTPσ(-/-) mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ(-/-) mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.

  14. Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

    PubMed

    Jordheim, Lars Petter; Cros-Perrial, Emeline; Matera, Eva-Laure; Bouledrak, Karima; Dumontet, Charles

    2014-10-01

    Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

  15. Self-Assembled Modified Soy Protein/Dextran Nanogel Induced by Ultrasonication as a Delivery Vehicle for Riboflavin.

    PubMed

    Jin, Bei; Zhou, Xiaosong; Li, Xiangzhong; Lin, Weiqin; Chen, Guangbin; Qiu, Riji

    2016-01-01

    A simple and green approach was developed to produce a novel nanogel via self-assembly of modified soy protein and dextran, to efficiently deliver riboflavin. First, modified soy protein was prepared by heating denaturation at 60 °C for 30 min or Alcalase hydrolysis for 40 min. Second, modified soy protein was mixed with dextran and ultrasonicated for 70 min so as to assemble nanogels. The modified soy protein-dextran nanogels were characterized by Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and ζ-potential studies to confirm the formation of NGs. Transmission electron microscopy (TEM) revealed the NGs to be spherical with core-shell structures, in the range of 32-40 nm size. The nanogels were stable against various environmental conditions. Furthermore, the particle size of the nanogels hardly changed with the incorporation of riboflavin. The encapsulation efficiency of nanogels was found to be up to 65.9% at a riboflavin concentration of 250 μg/mL. The nanogels exhibited a faster release in simulated intestine fluid (SIF) compared with simulated gastric fluid (SGF). From the results obtained it can be concluded that modified soy protein-dextran nanogels can be considered a promising carrier for drugs and other bioactive molecule delivery purposes. PMID:26999081

  16. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  17. ConPlex: a server for the evolutionary conservation analysis of protein complex structures.

    PubMed

    Choi, Yoon Sup; Han, Seong Kyu; Kim, Jinho; Yang, Jae-Seong; Jeon, Jouhyun; Ryu, Sung Ho; Kim, Sanguk

    2010-07-01

    Evolutionary conservation analyses are important for the identification of protein-protein interactions. For protein complex structures, sequence conservation has been applied to determine protein oligomerization states, to characterize native interfaces from non-specific crystal contacts, and to discriminate near-native structures from docking artifacts. However, a user-friendly web-based service for evolutionary conservation analysis of protein complexes has not been available. Therefore, we developed ConPlex (http://sbi.postech.ac.kr/ConPlex/) a web application that enables evolutionary conservation analyses of protein interactions within protein quaternary structures. Users provide protein complex structures; ConPlex automatically identifies protein interfaces and carries out evolutionary conservation analyses for the interface regions. Moreover, ConPlex allows the results of the residue-specific conservation analysis to be displayed on the protein complex structure and provides several options to customize the display output to fit each user's needs. We believe that ConPlex offers a convenient platform to analyze protein complex structures based on evolutionary conservation of protein-protein interface residues.

  18. Determination of disulfide array and subunit structure of taste-modifying protein, miraculin.

    PubMed

    Igeta, H; Tamura, Y; Nakaya, K; Nakamura, Y; Kurihara, Y

    1991-09-20

    The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity. PMID:1911854

  19. Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities.

    PubMed

    Ríos Quiroz, Anacelia; Québatte, Gabriela; Stump, Fabian; Finkler, Christof; Huwyler, Joerg; Schmidt, Roland; Mahler, Hanns-Christian; Koulov, Atanas V; Adler, Michael

    2015-06-16

    Although light obscuration is the "gold standard" for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 μm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 μm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements.

  20. Approaches to assessment of the allergenic potential of novel proteins in food from genetically modified crops.

    PubMed

    Kimber, Ian; Dearman, Rebecca J

    2002-07-01

    The safety assessment of food derived from genetically modified plants continues to attract considerable attention. Among the important issues that need to be considered is whether the products of novel genes introduced into crop plants will have the potential to induce allergic sensitization or to elicit allergic disease. Hierarchical approaches to allergenicity testing have been proposed, and these incorporate evaluation of the structural and sequence homology and serological identity of novel proteins with known allergens, measurement of resistance to proteolytic digestion, and assessment of allergenic potential using animal models. Accounts of these approaches are available elsewhere, and it is not the purpose of this article to provide a detailed critique of specific methods. Our intention is rather to look more broadly at the strategy for assessment of allergenic potential, the challenges such assessments pose for the practicing toxicologist, and how some of these might best be addressed.

  1. Nucleolin targeting AS1411 modified protein nanoparticle for antitumor drugs delivery.

    PubMed

    Wu, Jinhui; Song, Chenchen; Jiang, Chenxiao; Shen, Xin; Qiao, Qian; Hu, Yiqiao

    2013-10-01

    Over recent years, cell surface nucleolin as an anticancer target has attracted many researchers' attentions. To improve the antitumor efficacy, we developed a nucleolin targeted protein nanoparticle (NTPN) delivery system in which human serum albumin (HSA) was used as drug carrier and a DNA aptamer named AS1411, which had high affinity to nucleolin, was used as a bullet. The HSA nanoparticles (NPs-PTX) were fabricated by a novel self-assembly method and then modified with AS1411 (Apt-NPs-PTX). The resulted Apt-NPs-PTX were spherical. Compared with NPs-PTX, the uptake of Apt-NPs-PTX displayed a significant increase in MCF-7 cells while there was a decrease in nontumor cell lines such as MCF-10A and 3T3 cells. In a cytotoxic study, Apt-NPs-PTX displayed an enhanced cytotoxicity in MCF-7 tumor cells while there was almost no cytotoxicity in MCF-10A cells. Endostatin, a nucleolin inhibitor, could significantly decrease the internalization of Apt-NPs-PTX, suggesting nucleolin mediates the transmembrane process of Apt-NPs-PTX. Therefore, the AS1411 modified NTPN delivery system might be a promising targeted drug delivery system. PMID:23679916

  2. Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines

    PubMed Central

    Nacer, Adéla; Carapau, Daniel; Mitchell, Robert; Meltzer, Abby; Shaw, Alan; Frevert, Ute; Nardin, Elizabeth H

    2013-01-01

    Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a TLR5 agonist, was found to elicit antibody mediated protective immunity in our previous murine studies. To better understand IN elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DC) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DC localized beneath the epithelium and within the GC T cell area. We detected microfold (M) cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DC in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT APC populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria. PMID:23820750

  3. Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit.

    PubMed

    Theerasilp, S; Kurihara, Y

    1988-08-15

    The taste-modifying protein, miraculin, has the unusual property of modifying a sour taste into a sweet taste. Previous attempts to isolate miraculin from deeply colored alkaline extracts of the miracle fruit were unsuccessful. We found that miraculin is extracted with 0.5 M NaCl solution. The extracted solution is colorless and shows the strong sweet-inducing activity. Miraculin was purified from the extracted solution by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose affinity chromatography. The purified miraculin thus obtained gave a single sharp peak in reverse phase high performance liquid chromatography, indicating that it is highly pure. The sample also gave a single band having molecular weight 28,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was much lower than the values reported previously (40,000-48,000). The amino acid composition of the purified miraculin was determined. Sequence analysis of the purified miraculin indicated that it is composed of a pure single polypeptide and identified 20 amino-terminal amino acids. The purified miraculin contained as much as 13.9% of sugars, which consisted of glucosamine, mannose, galactose, xylose, and fucose in a molar ratio of 3.03:3.00:0.69:0.96:2.12. PMID:3403544

  4. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

    PubMed Central

    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.

    2016-01-01

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  5. Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

    PubMed

    Campos, Samuel K; Parrott, M Brandon; Barry, Michael A

    2004-06-01

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.

  6. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Kumar, Sugam; Aswal, V. K.

    2014-04-01

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  7. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    SciTech Connect

    Mehan, Sumit Kumar, Sugam Aswal, V. K.

    2014-04-24

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  8. Development, characterization and applications of electrodes modified with conductive polymers, ionic liquids and proteins

    NASA Astrophysics Data System (ADS)

    Tang, Yijun

    My research involves both fundamental studies and applications of the electrodes whose surfaces are chemically modified. Conductive polymers are one of the major materials that are used to modify electrode surfaces. The thorough understanding of the behavior of conductive polymers in ionic liquids is interesting and important as the ionic liquids are becoming promising solvents. With poly(vinyl ferrocene) as the model conductive polymer, electrochemical studies were performed in various ionic liquid electrolytes. A theoretical square model and dynamic equilibrium were proposed to describe the interaction between conductive polymers and ionic liquids when the electrons transferred between the electrode and electrolyte. These findings were applied to enable and accelerate the structure relaxation of conductive polymers so that the conductive polymers were capable of delivering peptides efficiently. Incorporation of metallic nanoparticles to the conductive polymer matrix entitled new properties to the conductive polymer, increasing conductivity and providing catalytic abilities. This modification on electrode surface might bring potential uses in gas sensing, energy storage, energy conversion, etc. Conductive polymer coated electrodes produced unique double layer in ionic liquids and a fundamental study of quantum charging help to understand the double layer properties. I also studied the application of surface modified electrodes in chemo- and biosensing. A nonregeneration protocol was created to save the cost and the time in analyzing interfacial binding activities and to prevent the potential of deterioration caused to biological ligands by the conventional regeneration. In the study of carbohydrate/protein interactions, a "click" chemical reaction was first used in constructing a carbohydrate-based biosensor, which was capable of detecting and analyzing proteins specifically and accurately. In another biosensor design, the hydrogen bonding between the template and

  9. Toluene 4-Monooxygenase and its Complex with Effector Protein T4moD

    SciTech Connect

    Bailey, Lucas J.; Fox, Brian G.

    2012-10-16

    Toluene 4-monooxygenase (T4MO) is a multiprotein diiron enzyme complex that catalyzes the regiospecific oxidation of toluene to p-cresol. Catalytic function requires the presence of a small protein, called the effector protein. Effector protein exerts substantial control on the diiron hydroxylase catalytic cycle through protein-protein interactions. High-resolution crystal structures of the stoichometric hydroxylase and effector protein complex described here reveal how protein-protein interactions and reduction of the diiron center produce an active site configuration poised for reaction with O{sub 2}. Further information from crystal structures of mutated isoforms of the hydroxylase and a peroxo adduct is combined with catalytic results to give a fuller picture of the geometry of the enzyme-substrate complex used for the high fidelity oxidation of hydrocarbon substrates.

  10. The orthopoxvirus 68-kilodalton ankyrin-like protein is essential for DNA replication and complete gene expression of modified vaccinia virus Ankara in nonpermissive human and murine cells.

    PubMed

    Sperling, Karin M; Schwantes, Astrid; Staib, Caroline; Schnierle, Barbara S; Sutter, Gerd

    2009-06-01

    Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus. We showed previously that 68k-ank is composed of ankyrin repeats and an F-box-like domain and forms an SCF ubiquitin ligase complex together with the cellular proteins Skp1a and Cullin-1. We now report that 68k-ank (MVA open reading frame 186R) is an essential factor for completion of the MVA intracellular life cycle in nonpermissive human and murine cells. Infection of mouse NIH 3T3 and human HaCaT cells with MVA with a deletion of the 68k-ank gene (MVA-Delta68k-ank) was characterized by an extensive reduction of viral intermediate RNA and protein, as well as late transcripts and drastically impaired late protein synthesis. Furthermore, infections with MVA-Delta68k-ank failed to induce the host protein shutoff that is characteristic of VACV infections. Although we demonstrated that proteasome function in general is essential for the completion of the MVA molecular life cycle, we found that a mutant 68k-ank protein with a deletion of the F-box-like domain was able to fully complement the deficiency of MVA-Delta68k-ank to express all classes of viral genes. Thus, our data demonstrate that the 68k-ank protein contains another critical domain that may function independently of SCF ubiquitin ligase complex formation, suggesting multiple activities of this interesting regulatory protein.

  11. Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

    PubMed

    Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

    2006-09-01

    An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

  12. Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function.

    PubMed

    Sangsuwan, Jiraporn; Wanichpakorn, Supreya; Kedjarune-Leggat, Ureporn

    2015-09-01

    The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC+TCTP, BIO-GIC and BIO-GIC+TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC+TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC+TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC+TCTP can promote osteoblast cells proliferation, differentiation and function.

  13. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine

    PubMed Central

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L.; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  14. KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling*

    PubMed Central

    DiStefano, Peter V.; Kuebel, Julia M.; Sarelius, Ingrid H.; Glading, Angela J.

    2014-01-01

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1. PMID:25320085

  15. Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection.

    PubMed

    Horká, Marie; Růzicka, Filip; Horký, Jaroslav; Holá, Veronika; Slais, Karel

    2006-09-01

    We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3-10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5x10(2) to 1x10(3) with on-column UV detection at 280 nm.

  16. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine.

    PubMed

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  17. Tuning structure of oppositely charged nanoparticle and protein complexes

    SciTech Connect

    Kumar, Sugam Aswal, V. K.; Callow, P.

    2014-04-24

    Small-angle neutron scattering (SANS) has been used to probe the structures of anionic silica nanoparticles (LS30) and cationic lyszyme protein (M.W. 14.7kD, I.P. ∼ 11.4) by tuning their interaction through the pH variation. The protein adsorption on nanoparticles is found to be increasing with pH and determined by the electrostatic attraction between two components as well as repulsion between protein molecules. We show the strong electrostatic attraction between nanoparticles and protein molecules leads to protein-mediated aggregation of nanoparticles which are characterized by fractal structures. At pH 5, the protein adsorption gives rise to nanoparticle aggregation having surface fractal morphology with close packing of nanoparticles. The surface fractals transform to open structures of mass fractal morphology at higher pH (7 and 9) on approaching isoelectric point (I.P.)

  18. Kinetics of protein-protein complex coacervation and biphasic release of salbutamol sulfate from coacervate matrix.

    PubMed

    Tiwari, Ananya; Bindal, Sonal; Bohidar, H B

    2009-01-12

    Turbidimetric titration was used to initiate associative intermolecular interactions between a pair of protein molecules, gelatin-A and gelatin-B, having complementary charges that led to pH-induced liquid-liquid phase separation and the formation of complex coacervate. The stoichiometric binding ratio was found to be [gelatin-A]/[gelatin-B]=3:2. The size of soluble intermolecular aggregates present in the supernatant exhibited interesting time-dependent coacervation because of residual electrostatic interactions. Dynamic light scattering and turbidity studies provided a systematic account of coacervation behavior. Rheology studies attributed the softening of the coacervate matrix to the presence of encapsulated salbutamol sulfate. The in vitro drug release kinetics was probed in simulated gastric fluid medium at physiological temperature (37 degrees C), which showed biphasic behavior. The initial release kinetics exhibited an exponential growth to saturation behavior, followed by a slower logarithmic release process.

  19. A 1 MDa protein complex containing critical components of the Escherichia coli divisome

    PubMed Central

    Trip, Erik N.; Scheffers, Dirk-Jan

    2015-01-01

    Cell division in bacteria is an essential process that is carried out at mid-cell by a group of cell division proteins referred to as the divisome. In Escherichia coli, over two dozen cell division proteins have been identified of which ten are essential. These division proteins localize sequentially and interdependently to the division site, after which constriction eventually produces two daughter cells. Various genetic and biochemical techniques have identified many interactions amongst cell division proteins, however the existence of the divisome as a large multi-protein complex has never been shown. Here, we identify a 1 MDa protein complex by native page that contains seven essential cell division proteins (FtsZ, ZipA, FtsK, FtsQ, FtsB, FtsL, and FtsN). The 1 MDa complex is present in rapidly dividing cells, but absent when cultures enter the stationary growth phase. Slight overexpression of the ftsQ D237N mutation that blocks cell division prevents formation of this 1 MDa complex. In cells depleted of FtsN, the 1 MDa complex is not assembled. Combined, our findings indicate that a large protein complex containing many different cell division proteins indeed exists. We note that this complex is very fragile and sensitive to the expression of tagged versions of FtsQ. PMID:26643979

  20. A Proteomic Strategy for Global Analysis of Plant Protein Complexes[W][OPEN

    PubMed Central

    Aryal, Uma K.; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C.; Szymanski, Daniel B.

    2014-01-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions. PMID:25293756

  1. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  2. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  3. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  4. Development of Cy5.5-Labeled Hydrophobically Modified Glycol Chitosan Nanoparticles for Protein Delivery

    NASA Astrophysics Data System (ADS)

    Chin, Amanda

    Therapeutic proteins are often highly susceptible to enzymatic degradation, thus restricting their in vivo stability. To overcome this limitation, delivery systems designed to promote uptake and reduce degradation kinetics have undergone a rapid shift from macro-scale systems to nanomaterial based carriers. Many of these nanomaterials, however, elicit immune responses and may have cytotoxic effects both in vitro and in vivo. The naturally derived polysaccharide chitosan has emerged as a promising biodegradable material and has been utilized for many biomedical applications; nevertheless, its function is often constrained by poor solubility. Glycol chitosan, a derivative of chitosan, can be hydrophobically modified to impart amphiphilic properties that enable the self-assembly into nanoparticles in aqueous media at neutral pH. This nanoparticle system has shown initial success as a therapeutic agent in several model cell culture systems, but little is known about its stability against enzymatic degradation. Therefore, the goal of this research was to investigate the resistance of hydrophobically modified glycol chitosan against enzyme-catalyzed degradation using an in vivo simulated system containing lysozyme. To synthesize the nanoparticles, hydrophobic cholanic acid was first covalently conjugated to glycol chitosan using of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Conjugates were purified by dialysis, lyophilized, and ultra-sonicated to form nanoparticles. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of 5beta-cholanic acid to the glycol chitosan. Particle size and stability over time were determined with dynamic light scattering (DLS), and particle morphology was evaluated by transmission electron microscopy (TEM). The average diameter of the nanoparticles was approximately 200 nm, which remained stable at 4°C for up to 10 days. Additionally, a near infrared fluorescent (NIRF) dye

  5. Modified diglycol-amides for actinide separation: solvent extraction and time-resolved laser fluorescence spectroscopy complexation studies

    SciTech Connect

    Wilden, A.; Modolo, G.; Lange, S.; Sadowski, F.; Bosbach, D.; Beele, B.B.; Panak, P.J.; Skerencak-Frech, A.; Geist, A.; Iqbal, M.; Verboom, W.

    2013-07-01

    In this work, the back-bone of the diglycolamide-structure of the TODGA extractant was modified by adding one or two methyl groups to the central methylene carbon-atoms. The influence of these structural modifications on the extraction behavior of trivalent actinides and lanthanides and other fission products was studied in solvent extraction experiments. The addition of methyl groups to the central methylene carbon atoms leads to reduced distribution ratios, also for Sr(II). This reduced extraction efficiency for Sr(II) is beneficial for process applications, as the co-extraction of Sr(II) can be avoided, resulting in an easier process design. The use of these modified diglycol-amides in solvent extraction processes is discussed. Furthermore, the complexation of Cm(III) and Eu(III) to the ligands was studied using Time-Resolved-Laser-Fluorescence-Spectroscopy (TRLFS). The complexes were characterized by slope analysis and conditional stability constants were determined.

  6. The modified Hospital Elder Life Program: adapting a complex intervention for feasibility and scalability in a surgical setting.

    PubMed

    Chen, Cheryl Chia-Hui; Saczynski, Jane; Inouye, Sharon K

    2014-05-01

    The purpose of this article is to provide the rationale and methods for adapting the Hospital Elder Life Program (HELP). The HELP is a complex intervention that has been shown to reduce rates of delirium and functional decline. However, modification of the program may be required to meet local circumstances and specialized populations. We selected three key elements based on our prior work and the concept of shared risk factors and modified the HELP to include only three shared risk factors (functional, nutritional, and cognitive status) that were targeted by three nursing protocols: early mobilization, oral and nutritional assistance, and orienting communication. These protocols were adapted, refined, and pilot-tested for feasibility and efficacy. We hope by reporting the rationale and protocols for the modified HELP, we will advance the field for others adapting evidence-based, complex nursing interventions.

  7. The modified Hospital Elder Life Program: adapting a complex intervention for feasibility and scalability in a surgical setting.

    PubMed

    Chen, Cheryl Chia-Hui; Saczynski, Jane; Inouye, Sharon K

    2014-05-01

    The purpose of this article is to provide the rationale and methods for adapting the Hospital Elder Life Program (HELP). The HELP is a complex intervention that has been shown to reduce rates of delirium and functional decline. However, modification of the program may be required to meet local circumstances and specialized populations. We selected three key elements based on our prior work and the concept of shared risk factors and modified the HELP to include only three shared risk factors (functional, nutritional, and cognitive status) that were targeted by three nursing protocols: early mobilization, oral and nutritional assistance, and orienting communication. These protocols were adapted, refined, and pilot-tested for feasibility and efficacy. We hope by reporting the rationale and protocols for the modified HELP, we will advance the field for others adapting evidence-based, complex nursing interventions. PMID:24443887

  8. A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex.

    PubMed

    Hong, Ming; Zha, Lei; Fu, Wenliang; Zou, Minji; Li, Wuju; Xu, Donggang

    2012-02-01

    This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy/μl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas. PMID:22806847

  9. Protein-arginine methyltransferase 1 (PRMT1) methylates Ash2L, a shared component of mammalian histone H3K4 methyltransferase complexes.

    PubMed

    Butler, Jill S; Zurita-Lopez, Cecilia I; Clarke, Steven G; Bedford, Mark T; Dent, Sharon Y R

    2011-04-01

    Multiple enzymes and enzymatic complexes coordinately regulate the addition and removal of post-translational modifications on histone proteins. The oncoprotein Ash2L is a component of the mixed lineage leukemia (MLL) family members 1-4, Setd1A, and Setd1B mammalian histone H3K4 methyltransferase complexes and is essential to maintain global trimethylation of histone H3K4. However, regulation of these complexes at the level of expression and activity remains poorly understood. In this report, we demonstrate that Ash2L is methylated on arginine residues both in vitro and in cells. We found that both protein-arginine methyltransferases 1 and 5 methylate Arg-296 within Ash2L. These findings are the first to demonstrate that post-translational modifications occur on the Ash2L protein and provide a novel example of cross-talk between chromatin-modifying enzyme complexes. PMID:21285357

  10. Theory of polyelectrolyte adsorption on heterogeneously charged surfaces applied to soluble protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    de Vries, R.; Weinbreck, F.; de Kruif, C. G.

    2003-03-01

    Existing theoretical approaches to polymer adsorption on heterogeneous surfaces are applied to the problems of polyelectrolyte and polyampholyte adsorption on randomly charged surfaces. Also, analytical estimates are developed for the critical pH at which weakly charged polyelectrolytes and globular proteins start forming soluble complexes. Below a critical salt concentration, soluble complexes form "on the wrong side" of the protein isoelectric point due to the heterogeneity of the protein surface charge distribution. The analytical estimates are consistent with experimental data on soluble complexes in mixtures of gum arabic and whey protein isolate.

  11. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    PubMed Central

    Cannistraci, Carlo Vittorio; Abbas, Ahmed; Gao, Xin

    2015-01-01

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet's performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis. PMID:25619991

  12. Soliton solutions of the (2+1)-dimensional complex modified Korteweg-de Vries and Maxwell-Bloch equations

    NASA Astrophysics Data System (ADS)

    Yesmakhanova, K.; Bekova, G.; Shaikhova, G.; Myrzakulov, R.

    2016-08-01

    In this paper, we consider the (2+1)-dimensional complex modified Korteweg-de Vries and Maxwell-Bloch (cmKdVMB) equations. Lax pairs of cmKdVMB equations are presented. Using the Lax pair, we construct a Darboux transformation and namely one-fold transformations. The soliton solutions are obtained from the different “seeds” by using this Darboux transformation.

  13. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    NASA Astrophysics Data System (ADS)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  14. Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin.

    PubMed

    Yamashita, H; Theerasilp, S; Aiuchi, T; Nakaya, K; Nakamura, Y; Kurihara, Y

    1990-09-15

    A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed. PMID:2394746

  15. Clinical Role of Modified Seton Procedure and Coring Out for Treatment of Complex Anal Fistulas Associated With Hidradenitis Suppurativa

    PubMed Central

    Tokunaga, Yukihiko; Sasaki, Hirokazu

    2015-01-01

    A variety of techniques have been described to treat complex anal fistulas. When complex anal fistulas are associated with hidradenitis suppurativa, the treatment has to be appropriately tailored for the severity and distribution of the disease so as to remove the external fistula tract to prevent recurrence while ensuring fecal continence. Between 2007 and 2011, a total of 10 males (ranging in age from 32 to 54 years) complained of recurrent purulent discharge in the buttocks and thigh regions. The discharge had started about 12 to 18 months prior, and had increased progressively resulting in complex anal fistulas and hidradenitis suppurativa in the buttocks. They underwent surgical operation according to a modified seton procedure for complex anal fistulas and coring out for hidradenitis suppurativa. They were discharged from the hospital in 4 to 5 days, while the seton dropped spontaneously about 6 to 8 months after surgery. They have been well without any morbidities or recurrence. The present paper demonstrates that cases of complex anal fistulas associated with hidradenitis suppurativa can be successfully treated with a modified seton procedure and coring out of hidradenitis suppurativa. PMID:26414817

  16. Clinical Role of Modified Seton Procedure and Coring Out for Treatment of Complex Anal Fistulas Associated With Hidradenitis Suppurativa.

    PubMed

    Tokunaga, Yukihiko; Sasaki, Hirokazu

    2015-06-01

    A variety of techniques have been described to treat complex anal fistulas. When complex anal fistulas are associated with hidradenitis suppurativa, the treatment has to be appropriately tailored for the severity and distribution of the disease so as to remove the external fistula tract to prevent recurrence while ensuring fecal continence. Between 2007 and 2011, a total of 10 males (ranging in age from 32 to 54 years) complained of recurrent purulent discharge in the buttocks and thigh regions. The discharge had started about 12 to 18 months prior, and had increased progressively resulting in complex anal fistulas and hidradenitis suppurativa in the buttocks. They underwent surgical operation according to a modified seton procedure for complex anal fistulas and coring out for hidradenitis suppurativa. They were discharged from the hospital in 4 to 5 days, while the seton dropped spontaneously about 6 to 8 months after surgery. They have been well without any morbidities or recurrence. The present paper demonstrates that cases of complex anal fistulas associated with hidradenitis suppurativa can be successfully treated with a modified seton procedure and coring out of hidradenitis suppurativa.

  17. Production of unstable proteins through the formation of stable core complexes.

    PubMed

    Levy, Nicolas; Eiler, Sylvia; Pradeau-Aubreton, Karine; Maillot, Benoit; Stricher, François; Ruff, Marc

    2016-01-01

    Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes. PMID:26983699

  18. Production of unstable proteins through the formation of stable core complexes

    PubMed Central

    Levy, Nicolas; Eiler, Sylvia; Pradeau-Aubreton, Karine; Maillot, Benoit; Stricher, François; Ruff, Marc

    2016-01-01

    Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes. PMID:26983699

  19. Structural Basis for the Coevolution of a Viral RNA-Protein Complex

    SciTech Connect

    Chao,J.; Patskovsky, Y.; Almo, S.; Singer, R.

    2008-01-01

    The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved beta-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.

  20. Determination of the structure and morphology of gold nanoparticle-HSA protein complexes.

    PubMed

    Capomaccio, Robin; Jimenez, Isaac Ojea; Colpo, Pascal; Gilliland, Douglas; Ceccone, Giacomo; Rossi, François; Calzolai, Luigi

    2015-11-14

    We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems. PMID:26462441

  1. Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

    PubMed Central

    Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

    2013-01-01

    Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

  2. Protein Nanocages for Delivery and Release of Luminescent Ruthenium(II) Polypyridyl Complexes.

    PubMed

    Li, Xiao; Zhang, Yajie; Chen, Hong; Sun, Jian; Feng, Fude

    2016-09-01

    In this report, noncovalent encapsulation of hydrophobic ruthenium(II) polyridyl complexes, Ru(bpy)2dppz(2+) and Ru(phen)2dppz(2+), into apoferritin cavity was achieved with high loading contents by effective prevention of Ru complex-induced protein aggregation, without disruption of protein native architecture. The Ru-loaded luminescent nanocomposites have demonstrated improved water solubility, easy manipulation, reduced cytotoxicity, and enhanced cellular uptake as compared to the nontreated Ru complexes. PMID:27547981

  3. Understanding the nanoparticle-protein corona complexes using computational and experimental methods.

    PubMed

    Kharazian, B; Hadipour, N L; Ejtehadi, M R

    2016-06-01

    Nanoparticles (NP) have capability to adsorb proteins from biological fluids and form protein layer, which is called protein corona. As the cell sees corona coated NPs, the protein corona can dictate biological response to NPs. The composition of protein corona is varied by physicochemical properties of NPs including size, shape, surface chemistry. Processing of protein adsorption is dynamic phenomena; to that end, a protein may desorb or leave a surface vacancy that is rapidly filled by another protein and cause changes in the corona composition mainly by the Vroman effect. In this review, we discuss the interaction between NP and proteins and the available techniques for identification of NP-bound proteins. Also we review current developed computational methods for understanding the NP-protein complex interactions.

  4. Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.

    PubMed

    Lin, Miao-Fang; Williams, Christie; Murray, Michael V; Ropp, Philip A

    2005-02-25

    During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.

  5. GPCR: G protein complexes--the fundamental signaling assembly.

    PubMed

    Jastrzebska, Beata

    2013-12-01

    G protein coupled receptors (GPCR) constitute the largest group of cell surface receptors that transmit various signals across biological membranes through the binding and activation of heterotrimeric G proteins, which amplify the signal and activate downstream effectors leading to the biological responses. Thus, the first critical step in this signaling cascade is the interaction between receptor and its cognate G protein. Understanding this critical event at the molecular level is of high importance because abnormal function of GPCRs is associated with many diseases. Thus, these receptors are targets for drug development.

  6. A single Sec61-complex functions as a protein-conducting channel.

    PubMed

    Kalies, Kai-Uwe; Stokes, Vivica; Hartmann, Enno

    2008-12-01

    During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

  7. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes.

    PubMed

    Han, Kyungsook; Nepal, Chirag

    2007-05-01

    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.

  8. A sustainable slashing industry using biodegradable sizes from modified soy protein to replace petro-based poly(vinyl alcohol).

    PubMed

    Zhao, Yi; Zhao, Yuzhu; Xu, Helan; Yang, Yiqi

    2015-02-17

    Biodegradable sizing agents from triethanolamine (TEA) modified soy protein could substitute poly(vinyl alcohol)(PVA) sizes for high-speed weaving of polyester and polyester/cotton yarns to substantially decrease environmental pollution and impel sustainability of textile industry. Nonbiodegradable PVA sizes are widely used and mainly contribute to high chemical oxygen demand (COD) in textile effluents. It has not been possible to effectively degrade, reuse or replace PVA sizes so far. Soy protein with good biodegradability showed potential as warp sizes in our previous studies. However, soy protein sizes lacked film flexibility and adhesion for required high-speed weaving. Additives with multiple hydroxyl groups, nonlinear molecule, and electric charge could physically modify secondary structure of soy protein and lead to about 23.6% and 43.3% improvement in size adhesion and ability of hair coverage comparing to unmodified soy protein. Industrial weaving results showed TEA-soy protein had relative weaving efficiency 3% and 10% higher than PVA and chemically modified starch sizes on polyester/cotton fabrics, and had relative weaving efficiency similar to PVA on polyester fabrics, although with 3- 6% lower add-on. In addition, TEA-soy sizes had a BOD5/COD ratio of 0.44, much higher than 0.03 for PVA, indicating that TEA-soy sizes were easily biodegradable in activated sludge.

  9. Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

    PubMed

    Lau, Julia B; Stork, Simone; Moog, Daniel; Schulz, Julian; Maier, Uwe G

    2016-04-01

    Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein-protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD-related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids.

  10. Folding and self-assembly of a small protein complex

    PubMed Central

    Sieradzan, Adam K.; Liwo, Adam; Hansmann, Ulrich H.E.

    2012-01-01

    The synthetic homotetrameric ββα (BBAT1) protein possesses a stable quaternary structure with a ββα fold. Because of its small size (a total of 84 residues), the homotetramer is an excellent model system with which to study the self-assembly and protein-protein interactions. We find from replica exchange molecular dynamics simulations with the coarse-grain UNRES force field that the folding and association pathway consists of three well-separated steps, where that association to a tetramer precedes and facilitates folding of the four chains. At room temperature the tetramer exists in an ensemble of diverse structures. The crystal structure becomes energetically favored only when the molecule is put in a dense and crystal-like environment. The observed picture of folding promoted by association may mirror the mechanism according to which intrinsically unfolded proteins assume their functional structure. PMID:24039552

  11. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  12. From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

    PubMed

    Hiwasa-Tanase, Kyoko; Hirai, Tadayoshi; Kato, Kazuhisa; Duhita, Narendra; Ezura, Hiroshi

    2012-03-01

    The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use. PMID:22160133

  13. Aggregate structure and effect of phthalic anhydride modified soy protein on the mechanical properties of styrene-butadiene copolymer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aggregate structure of phthalic anhydride (PA) modified soy protein isolate (SPI) was investigated by estimating its fractal dimension from the equilibrated dynamic strain sweep experiments. The estimated fractal dimensions of the filler aggregates were less than 2, indicating that these partic...

  14. From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

    PubMed

    Hiwasa-Tanase, Kyoko; Hirai, Tadayoshi; Kato, Kazuhisa; Duhita, Narendra; Ezura, Hiroshi

    2012-03-01

    The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.

  15. Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides

    PubMed Central

    Tchigvintsev, Anatoli; Tchigvintsev, Dmitri; Flick, Robert; Popovic, Ana; Dong, Aiping; Xu, Xiaohui; Brown, Greg; Lu, Wenyun; Wu, Hong; Cui, Hong; Dombrowski, Ludmila; Joo, Jeong Chan; Beloglazova, Natalia; Min, Jinrong; Savchenko, Alexei; Caudy, Amy A.; Rabinowitz, Joshua D.; Murzin, Alexey G.; Yakunin, Alexander F.

    2013-01-01

    Summary Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning. PMID:24210219

  16. On the interconnection of stable protein complexes: inter-complex hubs and their conservation in Saccharomyces cerevisiae and Homo sapiens networks.

    PubMed

    Guerra, Concettina

    2015-01-01

    Protein complexes are key molecular entities that perform a variety of essential cellular functions. The connectivity of proteins within a complex has been widely investigated with both experimental and computational techniques. We developed a computational approach to identify and characterise proteins that play a role in interconnecting complexes. We computed a measure of inter-complex centrality, the crossroad index, based on disjoint paths connecting proteins in distinct complexes and identified inter-complex hubs as proteins with a high value of the crossroad index. We applied the approach to a set of stable complexes in Saccharomyces cerevisiae and in Homo sapiens. Just as done for hubs, we evaluated the topological and biological properties of inter-complex hubs addressing the following questions. Do inter-complex hubs tend to be evolutionary conserved? What is the relation between crossroad index and essentiality? We found a good correlation between inter-complex hubs and both evolutionary conservation and essentiality.

  17. Impacts of bridging complexation on the transport of surface-modified nanoparticles in saturated sand

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transport of polyacrylic acid capped cadmium telluride (CdTe) quantum dots (QDs) and carboxylate-modified latex (CML) nanoparticles (NPs) was studied in packed columns at various electrolyte concentrations and cation types. The breakthrough curves (BTCs) of QDs and CML NPs in acid-treated Accus...

  18. Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein.

    PubMed

    Ohkubo, Takayuki; Tamiya, Minoru; Abe, Keiko; Ishiguro, Masaji

    2015-01-01

    Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness. PMID:26010443

  19. Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein

    PubMed Central

    Ohkubo, Takayuki; Tamiya, Minoru; Abe, Keiko; Ishiguro, Masaji

    2015-01-01

    Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness. PMID:26010443

  20. Injectable, thermo-reversible and complex coacervate combination gels for protein drug delivery.

    PubMed

    Jin, Kwang-Mi; Kim, Yong-Hee

    2008-05-01

    Injectable and thermo-reversible physical combination gels were formed in aqueous solution by preparing complex coacervate with two oppositely charged biomacromolecules that composed of negatively charged chondroitin 6-sulfate and positively charged high molecular weight gelatin type A and co-formulating with a negative, thermo-sensitive polysaccharide, methylcellulose containing a salting-out salt, ammonium sulfate. The combination of complex coacervation and a thermo-reversible gel demonstrated synergistic effects on the complex coacervate formation the release rates of model proteins and in situ gel depot formation. Gels indicated sustained release patterns of the protein over 25 days with minimal initial bursts. Optimized novel in situ gel depot systems containing dual advantages of complex coacervation and temperature responsiveness demonstrated a potential for efficient protein drug delivery in terms of high protein loading, sustained protein release, ease of administration, an aqueous environment without toxic organic solvents, and a simple fabrication method.