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Sample records for substrate binding interactions

  1. Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

    Directory of Open Access Journals (Sweden)

    Jose L S Lopes

    Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

  2. Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri.

    Science.gov (United States)

    Balan, Andrea; Santacruz-Pérez, Carolina; Moutran, Alexandre; Ferreira, Luís Carlos Souza; Neshich, Goran; Gonçalves Barbosa, João Alexandre Ribeiro

    2008-02-01

    In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.

  3. Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

    Science.gov (United States)

    Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

    2017-10-13

    Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  5. Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs).

    Science.gov (United States)

    Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I; Hill, Christopher P

    2015-05-22

    The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)*

    Science.gov (United States)

    Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I.; Hill, Christopher P.

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. PMID:25833946

  7. TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

    Science.gov (United States)

    Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

    2016-06-01

    Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  8. PREFACE: Cell-substrate interactions Cell-substrate interactions

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    Gardel, Margaret; Schwarz, Ulrich

    2010-05-01

    One of the most striking achievements of evolution is the ability to build cellular systems that are both robust and dynamic. Taken by themselves, both properties are obvious requirements: robustness reflects the fact that cells are there to survive, and dynamics is required to adapt to changing environments. However, it is by no means trivial to understand how these two requirements can be implemented simultaneously in a physical system. The long and difficult quest to build adaptive materials is testimony to the inherent difficulty of this goal. Here materials science can learn a lot from nature, because cellular systems show that robustness and dynamics can be achieved in a synergetic fashion. For example, the capabilities of tissues to repair and regenerate are still unsurpassed in the world of synthetic materials. One of the most important aspects of the way biological cells adapt to their environment is their adhesive interaction with the substrate. Numerous aspects of the physiology of metazoan cells, including survival, proliferation, differentiation and migration, require the formation of adhesions to the cell substrate, typically an extracellular matrix protein. Adhesions guide these diverse processes both by mediating force transmission from the cell to the substrate and by controlling biochemical signaling pathways. While the study of cell-substrate adhesions is a mature field in cell biology, a quantitative biophysical understanding of how the interactions of the individual molecular components give rise to the rich dynamics and mechanical behaviors observed for cell-substrate adhesions has started to emerge only over the last decade or so. The recent growth of research activities on cell-substrate interactions was strongly driven by the introduction of new physical techniques for surface engineering into traditional cell biological work with cell culture. For example, microcontact printing of adhesive patterns was used to show that cell fate depends

  9. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  10. Functional relevance of AcrB Trimerization in pump assembly and substrate binding.

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    Wei Lu

    Full Text Available AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB.

  11. Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

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    Linenberger, Kimberly J.; Bretz, Stacey Lowery

    2015-01-01

    Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

  12. Substrate binding accelerates the conformational transitions and substrate dissociation in multidrug efflux transporter AcrB

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    Beibei eWang

    2015-04-01

    Full Text Available The tripartite efflux pump assembly AcrAB-TolC is the major multidrug resistance transporter in E. coli. The inner membrane transporter AcrB is a homotrimer, energized by the proton movement down the transmembrane electrochemical gradient. The asymmetric crystal structures of AcrB with three monomers in distinct conformational states (access (A, binding (B and extrusion (E support a functional rotating mechanism, in which each monomer of AcrB cycles among the three states in a concerted way. However, the relationship between the conformational changes during functional rotation and drug translocation has not been totally understood. Here, we explored the conformational changes of the AcrB homotrimer during the ABE→BEA transition in different substrate-binding states using targeted MD simulations. It was found that the dissociation of substrate from the distal binding pocket of B monomer is closely related to the concerted conformational changes in the translocation pathway, especially the side chain reorientation of Phe628 and Tyr327. A second substrate binding at the proximal binding pocket of A monomer evidently accelerates the conformational transitions as well as substrate dissociation in B monomer. The acceleration effect of the multi-substrate binding mode provides a molecular explanation for the positive cooperativity observed in the kinetic studies of substrate efflux and deepens our understanding of the functional rotating mechanism of AcrB.

  13. Interactions of Cannabinoids With Biochemical Substrates

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    Brian F Thomas

    2017-05-01

    Full Text Available Recent decades have seen much progress in the identification and characterization of cannabinoid receptors and the elucidation of the mechanisms by which derivatives of the Cannabis sativa plant bind to receptors and produce their physiological and psychological effects. The information generated in this process has enabled better understanding of the fundamental physiological and psychological processes controlled by the central and peripheral nervous systems and has fostered the development of natural and synthetic cannabinoids as therapeutic agents. A negative aspect of this decades-long effort is the proliferation of clandestinely synthesized analogs as recreational street drugs with dangerous effects. Currently, the interactions of cannabinoids with their biochemical substrates are extensively but inadequately understood, and the clinical application of derived and synthetic receptor ligands remains quite limited. The wide anatomical distribution and functional complexity of the cannabinoid system continue to indicate potential for both therapeutic and side effects, which offers challenges and opportunities for medicinal chemists involved in drug discovery and development.

  14. Molecular docking simulations provide insights in the substrate binding sites and possible substrates of the ABCC6 transporter.

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    Mohammad Jakir Hosen

    Full Text Available The human ATP-binding cassette family C member 6 (ABCC6 gene encodes an ABC transporter protein (ABCC6, primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE, an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS and leukotriene C4 (LTC4 were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3 was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.

  15. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    Science.gov (United States)

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  16. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

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    Caroline Lund Dahlberg

    Full Text Available Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  17. Structural Changes of Creatine Kinase upon Substrate Binding

    OpenAIRE

    Forstner, Michael; Kriechbaum, Manfred; Laggner, Peter; Wallimann, Theo

    1998-01-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzym...

  18. The productive cellulase binding capacity of cellulosic substrates.

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    Karuna, Nardrapee; Jeoh, Tina

    2017-03-01

    Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Selectivity of substrate binding and ionization of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

    Science.gov (United States)

    Luanloet, Thikumporn; Sucharitakul, Jeerus; Chaiyen, Pimchai

    2015-08-01

    2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the

  20. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

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    Ju-Chen Chia

    Full Text Available Phytochelatin synthase (PCS uses the substrates glutathione (GSH, γGlu-Cys-Gly and a cadmium (Cd-bound GSH (Cd∙GS2 to produce the shortest phytochelatin product (PC2, (γGlu-Cys2-Gly through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218 by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

  1. [Adenylate cyclase from rabbit heart: substrate binding site].

    Science.gov (United States)

    Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

    1981-08-01

    The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

  2. Structural changes of creatine kinase upon substrate binding.

    Science.gov (United States)

    Forstner, M; Kriechbaum, M; Laggner, P; Wallimann, T

    1998-08-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

  3. Structure of photosystem II and substrate binding at room temperature.

    Science.gov (United States)

    Young, Iris D; Ibrahim, Mohamed; Chatterjee, Ruchira; Gul, Sheraz; Fuller, Franklin; Koroidov, Sergey; Brewster, Aaron S; Tran, Rosalie; Alonso-Mori, Roberto; Kroll, Thomas; Michels-Clark, Tara; Laksmono, Hartawan; Sierra, Raymond G; Stan, Claudiu A; Hussein, Rana; Zhang, Miao; Douthit, Lacey; Kubin, Markus; de Lichtenberg, Casper; Long Vo, Pham; Nilsson, Håkan; Cheah, Mun Hon; Shevela, Dmitriy; Saracini, Claudio; Bean, Mackenzie A; Seuffert, Ina; Sokaras, Dimosthenis; Weng, Tsu-Chien; Pastor, Ernest; Weninger, Clemens; Fransson, Thomas; Lassalle, Louise; Bräuer, Philipp; Aller, Pierre; Docker, Peter T; Andi, Babak; Orville, Allen M; Glownia, James M; Nelson, Silke; Sikorski, Marcin; Zhu, Diling; Hunter, Mark S; Lane, Thomas J; Aquila, Andy; Koglin, Jason E; Robinson, Joseph; Liang, Mengning; Boutet, Sébastien; Lyubimov, Artem Y; Uervirojnangkoorn, Monarin; Moriarty, Nigel W; Liebschner, Dorothee; Afonine, Pavel V; Waterman, David G; Evans, Gwyndaf; Wernet, Philippe; Dobbek, Holger; Weis, William I; Brunger, Axel T; Zwart, Petrus H; Adams, Paul D; Zouni, Athina; Messinger, Johannes; Bergmann, Uwe; Sauter, Nicholas K; Kern, Jan; Yachandra, Vittal K; Yano, Junko

    2016-12-15

    Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn 4 CaO 5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S 0 to S 4 ), in which S 1 is the dark-stable state and S 3 is the last semi-stable state before O-O bond formation and O 2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S 1 ), two-flash illuminated (2F; S 3 -enriched), and ammonia-bound two-flash illuminated (2F-NH 3 ; S 3 -enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S 1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn 4 CaO 5 cluster in the S 2 and S 3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

  4. Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

    Science.gov (United States)

    Carland, Jane E; Thomas, Michael; Mostyn, Shannon N; Subramanian, Nandhitha; O'Mara, Megan L; Ryan, Renae M; Vandenberg, Robert J

    2018-03-21

    Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT Aa , and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuT Aa , contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

  5. Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

    Directory of Open Access Journals (Sweden)

    M.H. Tomassi

    2010-01-01

    Full Text Available The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly, both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39 were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex of the double mutations (∆∆G‡xy is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y. This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

  6. Characterization of the interdependency between residues that bind the substrate in a beta-glycosidase.

    Science.gov (United States)

    Tomassi, M H; Rozenfeld, J H K; Gonçalves, L M; Marana, S R

    2010-01-01

    The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the beta-glycosidase from Spodoptera frugiperda (Sfbetagly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfbetagly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl beta-galactoside and p-nitrophenyl beta-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfbetagly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ESdouble dagger (enzyme-transition state complex) of the double mutations (Gdouble daggerxy) is not the sum of the effects resulting from the single mutations (Gdouble daggerx and Gdouble daggery). This difference in Gdouble dagger indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in Gdouble daggerxy. Crystallographic data on beta-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

  7. Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements.

    Science.gov (United States)

    Liu, Shu; Bolger, Joshua K; Kirkland, Lindsay O; Premnath, Padmavathy N; McInnes, Campbell

    2010-12-17

    An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been

  8. RanBP3 influences interactions between CRM1 and its nuclear protein export substrates

    OpenAIRE

    Englmeier, Ludwig; Fornerod, Maarten; Bischoff, F. Ralf; Petosa, Carlo; Mattaj, Iain W.; Kutay, Ulrike

    2001-01-01

    We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1–export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mec...

  9. Binding Thermodynamics of Ferredoxin:NADP+ Reductase: Two Different Protein Substrates and One Energetics

    Science.gov (United States)

    Martínez-Júlvez, Marta; Medina, Milagros; Velázquez-Campoy, Adrián

    2009-01-01

    Abstract The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP+ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His299 FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics. PMID:19527656

  10. Biochemistry students' ideas about how an enzyme interacts with a substrate.

    Science.gov (United States)

    Linenberger, Kimberly J; Bretz, Stacey Lowery

    2015-01-01

    Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.

  11. Binding energies of hypernuclei and hypernuclear interactions

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R. [Argonne National Lab., IL (United States)]|[Univ. of Illinois, Chicago, IL (United States). Dept. of Physics; Murali, S.; Usmani, Q.N. [Jamia Millia Islamia, New Delhi (India). Dept. of Physics

    1996-05-01

    In part 1 the effect of nuclear core dynamics on the binding energies of {Lambda} hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the {Lambda} single-particle energy in terms of basic {Lambda}-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body {Lambda}NN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei.

  12. Binding energies of hypernuclei and hypernuclear interactions

    International Nuclear Information System (INIS)

    Bodmer, A.R.; Univ. of Illinois, Chicago, IL; Murali, S.; Usmani, Q.N.

    1996-01-01

    In part 1 the effect of nuclear core dynamics on the binding energies of Λ hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the Λ single-particle energy in terms of basic Λ-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body ΛNN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei

  13. Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.

    2000-01-01

    Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. in particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24...... for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein...

  14. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  15. Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

    NARCIS (Netherlands)

    Wolters, Justina C.; Berntsson, Ronnie P-A.; Gul, Nadia; Karasawa, Akira; Thunnissen, Andy-Mark W. H.; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein

  16. Substrate binding to SGLT1 investigated by single molecule force spectroscopy

    International Nuclear Information System (INIS)

    Neundlinger, I. J.

    2010-01-01

    D-glucose serves as one of the most important fuels in various organism due to its fundamental role in ATP-, protein and lipid synthesis. Thus, sustaining glucose homeostasis is a crucial issue of life as disorders can cause severe malfunctions such as glucose-galactose-malabsorbtion (GGM). Sodium-glucose co-transporter, SGLTs, especially the high affinity transporter SGLT1, play a crucial role in accumulation of glucose in the cell as they facilitate transport of the sugar into the cytoplasma across the cell membrane by a Na+-electrochemical potential. Even recently, members of the SGLT transporter family have become a therapeutic target for the treatment of hyperglycemia in type 2 diabetes. Hence, it is of particular importance to gain insights on the dynamic behavior of SGLTs during substrate binding and transport across the cell membrane on the single molecular level. In the present study, the Atomic Force Microscope (AFM) was employed to investigate the dynamic properties of the sodium-glucose co-transporter SGLT1 upon substrate binding under nearly physiological conditions. Hereto, new glucose derivatives were synthesized in order to probe the recognition efficiency of these molecules to SGLT1 embedded in the plasma membrane of living cells. A well established coupling protocol was used to covalently link (i) amino-modified D-glucose owning a conserved pyranose ring, (ii) 1-thio-β-D-glucose having a sulphur atom at C1 of the pyranose ring and (iii) the competitive inhibitor phlorizin to the AFM tip via poly(ethylene)glycol (PEG)-tether using different functional end groups and varying lengths. Binding characteristics, e.g. binding probability, interaction forces, influence of substances (glucose, phlorizin, sodium) and of molecule-linker compounds were obtained by performing single molecular recognition force spectroscopy (SMRFS) measurements. Moreover, temperature controlled radioactive binding/transport assays and SMRFS experiments yielded insights into

  17. Development of the Enzyme-Substrate Interactions Concept Inventory

    Science.gov (United States)

    Bretz, Stacey Lowery; Linenberger, Kimberly J.

    2012-01-01

    Enzyme function is central to student understanding of multiple topics within the biochemistry curriculum. In particular, students must understand how enzymes and substrates interact with one another. This manuscript describes the development of a 15-item Enzyme-Substrate Interactions Concept Inventory (ESICI) that measures student understanding…

  18. A Correlation between the Activity of Candida antarctica Lipase B and Differences in Binding Free Energies of Organic Solvent and Substrate

    DEFF Research Database (Denmark)

    Banik, Sindrila Dutta; Nordblad, Mathias; Woodley, John

    2016-01-01

    in an inhibitory effect which is also confirmed by the binding free energies for the solvent and substrate molecules estimated from the simulations. Consequently, the catalytic activity of CALB decreases in polar solvents. This effect is significant, and CALB is over 10 orders of magnitude more active in nonpolar...... of the enzyme may be ascribed to binding of solvent molecules to the enzyme active site region and the solvation energy of substrate molecules in the different solvents. Polar solvent molecules interact strongly with CALB and compete with the substrate to bind to the active site region, resulting...

  19. Decoding P4-ATPase substrate interactions.

    Science.gov (United States)

    Roland, Bartholomew P; Graham, Todd R

    Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca 2+ , a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.

  20. Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

    2008-01-01

    Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft with a charact......Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft...... will address surface sites in both barley alpha-amylase 1 and in the related isozyme 2....

  1. Enzyme-substrate binding landscapes in the process of nitrile biodegradation mediated by nitrile hydratase and amidase.

    Science.gov (United States)

    Zhang, Yu; Zeng, Zhuotong; Zeng, Guangming; Liu, Xuanming; Chen, Ming; Liu, Lifeng; Liu, Zhifeng; Xie, Gengxin

    2013-08-01

    The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme-substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and L-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme's binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.

  2. Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA.

    Directory of Open Access Journals (Sweden)

    Justina C Wolters

    2010-04-01

    Full Text Available The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC was characterized.The binding of glycine betaine to purified OpuA and OpuAC (K(D = 4-6 microM did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 A and closed-liganded (2.3 A conformation. The binding pocket is formed by three tryptophans (Trp-prism coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.

  3. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

  4. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  5. Multifunctionality is affected by interactions between green roof plant species, substrate depth, and substrate type.

    Science.gov (United States)

    Dusza, Yann; Barot, Sébastien; Kraepiel, Yvan; Lata, Jean-Christophe; Abbadie, Luc; Raynaud, Xavier

    2017-04-01

    Green roofs provide ecosystem services through evapotranspiration and nutrient cycling that depend, among others, on plant species, substrate type, and substrate depth. However, no study has assessed thoroughly how interactions between these factors alter ecosystem functions and multifunctionality of green roofs. We simulated some green roof conditions in a pot experiment. We planted 20 plant species from 10 genera and five families (Asteraceae, Caryophyllaceae, Crassulaceae, Fabaceae, and Poaceae) on two substrate types (natural vs. artificial) and two substrate depths (10 cm vs. 30 cm). As indicators of major ecosystem functions, we measured aboveground and belowground biomasses, foliar nitrogen and carbon content, foliar transpiration, substrate water retention, and dissolved organic carbon and nitrates in leachates. Interactions between substrate type and depth strongly affected ecosystem functions. Biomass production was increased in the artificial substrate and deeper substrates, as was water retention in most cases. In contrast, dissolved organic carbon leaching was higher in the artificial substrates. Except for the Fabaceae species, nitrate leaching was reduced in deep, natural soils. The highest transpiration rates were associated with natural soils. All functions were modulated by plant families or species. Plant effects differed according to the observed function and the type and depth of the substrate. Fabaceae species grown on natural soils had the most noticeable patterns, allowing high biomass production and high water retention but also high nitrate leaching from deep pots. No single combination of factors enhanced simultaneously all studied ecosystem functions, highlighting that soil-plant interactions induce trade-offs between ecosystem functions. Substrate type and depth interactions are major drivers for green roof multifunctionality.

  6. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

    Science.gov (United States)

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-01

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

  7. Functional Diversity of Tandem Substrate-Binding Domains in ABC Transporters from Pathogenic Bacteria

    NARCIS (Netherlands)

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Vujicic - Zagar, Andreja; Guskov, Albert; Slotboom, Dirk-Jan; Poolman, Bert

    2013-01-01

    The ATP-binding cassette (ABC) transporter GInPQ is an essential uptake system for amino acids in gram-positive pathogens and related nonpathogenic bacteria. The transporter has tandem substrate-binding domains (SBDs) fused to each transmembrane domain, giving rise to four SBDs per functional

  8. Substrate Binding Induces Domain Movements in Orotidine 5'-Monophosphate Decarboxylase

    DEFF Research Database (Denmark)

    Harris, Pernille Hanne; Poulsen, Jens-Christian Navarro; Jensen, Kaj Frank

    2002-01-01

    ); here we present the 2.5 Å structure of the uncomplexed apo enzyme, determined from twinned crystals. A structural analysis and comparison of the two structures of the E. coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12° around a hinge...

  9. Catalytic Efficiency of Basidiomycete Laccases: Redox Potential versus Substrate-Binding Pocket Structure

    Directory of Open Access Journals (Sweden)

    Olga A. Glazunova

    2018-04-01

    Full Text Available Laccases are copper-containing oxidases that catalyze a one-electron abstraction from various phenolic and non-phenolic compounds with concomitant reduction of molecular oxygen to water. It is well-known that laccases from various sources have different substrate specificities, but it is not completely clear what exactly provides these differences. The purpose of this work was to study the features of the substrate specificity of four laccases from basidiomycete fungi Trametes hirsuta, Coriolopsis caperata, Antrodiella faginea, and Steccherinum murashkinskyi, which have different redox potentials of the T1 copper center and a different structure of substrate-binding pockets. Enzyme activity toward 20 monophenolic substances and 4 phenolic dyes was measured spectrophotometrically. The kinetic parameters of oxidation of four lignans and lignan-like substrates were determined by monitoring of the oxygen consumption. For the oxidation of the high redox potential (>700 mV monophenolic substrates and almost all large substrates, such as phenolic dyes and lignans, the redox potential difference between the enzyme and the substrate (ΔE played the defining role. For the low redox potential monophenolic substrates, ΔE did not directly influence the laccase activity. Also, in the special cases, the structure of the large substrates, such as dyes and lignans, as well as some structural features of the laccases (flexibility of the substrate-binding pocket loops and some amino acid residues in the key positions affected the resulting catalytic efficiency.

  10. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

    Directory of Open Access Journals (Sweden)

    Cédric Grauffel

    Full Text Available Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs. In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p. To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG, hNaa10p/AcCoA/substrate (substrate=MLG, EEE. Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic, hNaa20p (hydrophobic/basic, hNaa30p (basic and hNaa50p (hydrophobic. We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide

  11. Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

    Science.gov (United States)

    Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

    2018-05-14

    The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

  12. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    Science.gov (United States)

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-07-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

  13. Insulin receptor substrate-3, interacting with Bcl-3, enhances p50 NF-{kappa}B activity

    Energy Technology Data Exchange (ETDEWEB)

    Kabuta, Tomohiro [Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657 (Japan); Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502 (Japan); Hakuno, Fumihiko; Cho, Yoshitake; Yamanaka, Daisuke; Chida, Kazuhiro [Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657 (Japan); Asano, Tomoichiro [Graduate School of Biomedical Science, Hiroshima University, Hiroshima 734-8551 (Japan); Wada, Keiji [Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502 (Japan); Takahashi, Shin-Ichiro, E-mail: atkshin@mail.ecc.u-tokyo.ac.jp [Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657 (Japan)

    2010-04-09

    The insulin receptor substrate (IRS) proteins are major substrates of both insulin receptor and insulin-like growth factor (IGF)-I receptor tyrosine kinases. Previously, we reported that IRS-3 is localized to both cytosol and nucleus, and possesses transcriptional activity. In the present study, we identified Bcl-3 as a novel binding protein to IRS-3. Bcl-3 is a nuclear protein, which forms a complex with the homodimer of p50 NF-{kappa}B, leading to enhancement of transcription through p50 NF-{kappa}B. We found that Bcl-3 interacts with the pleckstrin homology domain and the phosphotyrosine binding domain of IRS-3, and that IRS-3 interacts with the ankyrin repeat domain of Bcl-3. In addition, IRS-3 augmented the binding activity of p50 to the NF-{kappa}B DNA binding site, as well as the tumor necrosis factor (TNF)-{alpha}-induced transcriptional activity of NF-{kappa}B. Lastly, IRS-3 enhanced NF-{kappa}B-dependent anti-apoptotic gene induction and consequently inhibited TNF-{alpha}-induced cell death. This series of results proposes a novel function for IRS-3 as a transcriptional regulator in TNF-{alpha} signaling, distinct from its function as a substrate of insulin/IGF receptor kinases.

  14. Cell In Situ Zymography: Imaging Enzyme-Substrate Interactions.

    Science.gov (United States)

    Chhabra, Aastha; Rani, Vibha

    2017-01-01

    Zymography has long been used for the detection of substrate-specific enzyme activity. In situ zymography (ISZ), an adaptation from the conventional substrate zymography, is a widely employed technique useful for the detection, localization, and estimation of enzyme-substrate interactions in tissues. Here, we describe a protocol to detect 'in position' matrix metalloproteinase (MMP) activity in cells utilizing H9c2 cardiomyoblasts as a model. This technique is primarily adopted from the method used for histological sections and is termed as 'Cell in situ Zymography'. It is a simple, sensitive, and quantifiable methodology to assess the functional activity of an enzyme 'on site/in position' in cell culture.

  15. Interfacial interactions between calcined hydroxyapatite nanocrystals and substrates.

    Science.gov (United States)

    Okada, Masahiro; Furukawa, Keiko; Serizawa, Takeshi; Yanagisawa, Yoshihiko; Tanaka, Hidekazu; Kawai, Tomoji; Furuzono, Tsutomu

    2009-06-02

    Interfacial interactions between calcined hydroxyapatite (HAp) nanocrystals and surface-modified substrates were investigated by measuring adsorption behavior and adhesion strength with a quartz crystal microbalance (QCM) and a contact-mode atomic force microscope (AFM), respectively. The goal was to develop better control of HAp-nanocrystal coatings on biomedical materials. HAp nanocrystals with rodlike or spherical morphology were prepared by a wet chemical process followed by calcination at 800 degrees C with an antisintering agent to prevent the formation of sintered polycrystals. The substrate surface was modified by chemical reaction with a low-molecular-weight compound, or graft polymerization with a functional monomer. QCM measurement showed that the rodlike HAp nanocrystals adsorbed preferentially onto anionic COOH-modified substrates compared to cationic NH2- or hydrophobic CH3-modified substrates. On the other hand, the spherical nanocrystals adsorbed onto NH2- and COOH-modified substrates, which indicates that the surface properties of the HAp nanocrystals determined their adsorption behavior. The adhesion strength, which was estimated from the force required to move the nanocrystal in contact-mode AFM, on a COOH-grafted substrate prepared by graft polymerization was almost 9 times larger than that on a COOH-modified substrate prepared by chemical reaction with a low-molecular-weight compound, indicating that the long-chain polymer grafted on the substrate mitigated the surface roughness mismatch between the nanocrystal and the substrate. The adhesion strength of the nanocrystal bonded covalently by the coupling reaction to a Si(OCH3)-grafted substrate prepared by graft polymerization was approximately 1.5 times larger than that when adsorbed on the COOH-grafted substrate.

  16. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  17. Substrate strain induced interaction of adatoms on W (110)

    Science.gov (United States)

    Kappus, W.

    1980-09-01

    The interaction of adatoms due to elastic strains created in an elastically isotropic substrate is investigated. For cases where the adatoms occupy sites with low symmetry, an angular dependent interaction results which falls off as s-3 at large distances. An exact expression is given for the long range interaction in terms of an anisotropy parameter of the force dipole tensor. The short range interaction is calculated by introducing a smooth cutoff. Interactions of adatoms on near neighbour sites on W (110) are given.

  18. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures

    Czech Academy of Sciences Publication Activity Database

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšík, Martin; Peclinovská, Lucie; Majer, Pavel; Stříšovský, Kvido

    2014-01-01

    Roč. 33, č. 20 (2014), s. 2408-2421 ISSN 0261-4189 R&D Projects: GA ČR GAP305/11/1886; GA MŠk(CZ) LK11206; GA MŠk LO1302; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : intramembrane protease * rhomboid family * rhomboid protease * structure * substrate recognition Subject RIV: CE - Biochemistry Impact factor: 10.434, year: 2014

  19. The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter

    NARCIS (Netherlands)

    Mulligan, Christopher; Geertsma, Eric R.; Severi, Emmanuele; Kelly, David J.; Poolman, Bert; Thomas, Gavin H.

    2009-01-01

    Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific

  20. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

    2016-06-13

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

  1. Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

    Science.gov (United States)

    Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

    2003-03-01

    Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

  2. Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Windahl, Michael Skovbo; Sim, Lyann

    2015-01-01

    Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably...... reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that αconfine high activity of LD to branched...... starch synthesis....

  3. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

    Czech Academy of Sciences Publication Activity Database

    Németh, E.; Körtvélyesi, T.; Kožíšek, Milan; Thulstrup, P. W.; Christensen, H. E. M.; Asaka, M. N.; Nagata, K.; Gyurcsik, B.

    2014-01-01

    Roč. 19, č. 8 (2014), s. 1295-1303 ISSN 0949-8257 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016; Seventh Framework Programme of the European Union(XE) FP7-312284 Institutional support: RVO:61388963 Keywords : binding affinity * calorimetry * zinc nuclease * substrate induced folding * protein engineering Subject RIV: CE - Biochemistry Impact factor: 2.538, year: 2014

  4. Binding of mouse immunoglobulin G to polylysine-coated glass substrate for immunodiagnosis

    Science.gov (United States)

    Vashist, Sandeep Kumar; Tewari, Rupinder; Bajpai, Ram Prakash; Bharadwaj, Lalit Mohan; Raiteri, Roberto

    2006-12-01

    We report a method for immobilizing mouse immunoglobulin G (IgG) on polylysine-coated glass substrate for immunodiagnostic applications. Mouse IgG molecules were immobilized on polylysine-coated glass substrate employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and protein A. The amino groups of the polylysine-coated glass slide were cross linked to the carboxyl groups of protein A employing EDC crosslinker. Protein A was employed as it binds to the constant Fc region of antibodies keeping their antigen binding sites on the variable F ab region free to bind to antigens. The qualitative analysis of surface immobilized mouse IgG was done by fluorescent microscopy employing fluorescein isothiocyanate (FITC) labeled mouse IgG molecules. The immobilization densities of protein A and mouse IgG were determined by 3, 3', 4, 4'-tetramethyl benzidine (TMB) substrate assay employing horse radish peroxidise labelled molecules and were found to be 130 +/- 17 ng/cm2 and 596 +/- 31 ng/cm2 respectively. The biomolecular coatings analyzed by atomic force microscopy (AFM) were found to be uniform.

  5. Binding properties of SUMO-interacting motifs (SIMs) in yeast.

    Science.gov (United States)

    Jardin, Christophe; Horn, Anselm H C; Sticht, Heinrich

    2015-03-01

    Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

  6. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

    2000-01-01

    A steady state kinetic investigation of the Pi activation of 5-phospho-D-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased...... the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas...... saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...

  7. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-01-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

  9. Correcting binding parameters for interacting ligand-lattice systems

    Science.gov (United States)

    Hervy, Jordan; Bicout, Dominique J.

    2017-07-01

    Binding of ligands to macromolecules is central to many functional and regulatory biological processes. Key parameters characterizing ligand-macromolecule interactions are the stoichiometry, inducing the number of ligands per macromolecule binding site, and the dissociation constant, quantifying the ligand-binding site affinity. Both these parameters can be obtained from analyses of classical saturation experiments using the standard binding equation that offers the great advantage of mathematical simplicity but becomes an approximation for situations of interest when a ligand binds and covers more than one single binding site on the macromolecule. Using the framework of car-parking problem with latticelike macromolecules where each ligand can cover simultaneously several consecutive binding sites, we showed that employing the standard analysis leads to underestimation of binding parameters, i.e., ligands appear larger than they actually are and their affinity is also greater than it is. Therefore, we have derived expressions allowing to determine the ligand size and true binding parameters (stoichiometry and dissociation constant) as a function of apparent binding parameters retrieved from standard saturation experiments.

  10. Regulatory site of inorganic pyrophosphatase. Interaction with substrate analogs

    International Nuclear Information System (INIS)

    Baikov, A.A.; Pavlov, A.R.; Avaeva, S.M.

    1986-01-01

    The effect of four PP 1 analogs with the structure PXP (X = N, C), phosphate, and the complex Cr(H 2 O) 4 PP 1 on the activity of inorganic pyrophosphatase from baker's yeast was studied over a wide range of substrate (Mg-PP 1 ) concentrations (lower limit 0.5 μM). The enzyme activity decreased in the presence of imidodiphosphate, hydroxymethane diphosphonate [PC(OH)P], and P 1 , and a double reciprocal plot of the rate of hydrolysis of Mg-PP 1 versus its concentration became linear. Small amounts of methane diphosphonate (PCP), ethane-1-hydroxy-1,1-diphosphonate (0.1-1μM), and Cr(H 2 O) 4 PP 1 (10 μM) activated the enzyme almost 2-fold by a competitive mechanism. The activation was due to an increase in the affinity of the protein for the activating Mg 2+ ion. Ultrafiltration showed that the pyrophosphatase molecule has 2.1 and 3.1 binding sites for PCP and PC(OHP)P, respectively. These results confirm the hypothesis that the enzyme contains a regulatory site whose occupation by PP 1 , P 1 , and substrate analogs increases the affinity of the protein for the activating metal

  11. Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

    Science.gov (United States)

    Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

    2014-05-01

    Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

  12. MNK Controls mTORC1:Substrate Association through Regulation of TELO2 Binding with mTORC1

    Directory of Open Access Journals (Sweden)

    Michael C. Brown

    2017-02-01

    Full Text Available The mechanistic target of rapamycin (mTOR integrates numerous stimuli and coordinates the adaptive response of many cellular processes. To accomplish this, mTOR associates with distinct co-factors that determine its signaling output. While many of these co-factors are known, in many cases their function and regulation remain opaque. The MAPK-interacting kinase (MNK contributes to rapamycin resistance in cancer cells. Here, we demonstrate that MNK sustains mTORC1 activity following rapamycin treatment and contributes to mTORC1 signaling following T cell activation and growth stimuli in cancer cells. We determine that MNK engages with mTORC1, promotes mTORC1 association with the phosphatidyl inositol 3′ kinase-related kinase (PIKK stabilizer, TELO2, and facilitates mTORC1:substrate binding. Moreover, our data suggest that DEPTOR, the endogenous inhibitor of mTOR, opposes mTORC1:substrate association by preventing TELO2:mTORC1 binding. Thus, MNK orchestrates counterbalancing forces that regulate mTORC1 enzymatic activity.

  13. A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

    Science.gov (United States)

    Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

    2016-11-01

    The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

  14. Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

    Energy Technology Data Exchange (ETDEWEB)

    Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita; Karkashon, Shay; Bonanno, Jeffrey B.; Poulos, Thomas L.; Yeh, Syun-Ru (Einstein); (UCI)

    2017-11-22

    Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

  15. Substrate interactions and promiscuity in a viral DNA packaging motor.

    Science.gov (United States)

    Aathavan, K; Politzer, Adam T; Kaplan, Ariel; Moffitt, Jeffrey R; Chemla, Yann R; Grimes, Shelley; Jardine, Paul J; Anderson, Dwight L; Bustamante, Carlos

    2009-10-01

    The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

  16. The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

    Science.gov (United States)

    Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

    2016-05-09

    The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity

  17. Configuration interaction calculations of positron binding to Be(3P )

    International Nuclear Information System (INIS)

    Bromley, M.W.J.; Mitroy, J.

    2006-01-01

    The configuration interaction method is applied to investigate the possibility of positron binding to the metastable beryllium (1s 2 2s2p 3 P ) state. The largest calculation obtained an estimated energy that was unstable by 0.00014 Hartree with respect to the Ps + Be + (2s) lowest dissociation channel. It is likely that positron binding to parent states with non-zero angular momentum is inhibited by centrifugal barriers

  18. Photosynthetic water oxidation: binding and activation of substrate waters for O-O bond formation.

    Science.gov (United States)

    Vinyard, David J; Khan, Sahr; Brudvig, Gary W

    2015-01-01

    Photosynthetic water oxidation occurs at the oxygen-evolving complex (OEC) of Photosystem II (PSII). The OEC, which contains a Mn4CaO5 inorganic cluster ligated by oxides, waters and amino-acid residues, cycles through five redox intermediates known as S(i) states (i = 0-4). The electronic and structural properties of the transient S4 intermediate that forms the O-O bond are not well understood. In order to gain insight into how water is activated for O-O bond formation in the S4 intermediate, we have performed a detailed analysis of S-state dependent substrate water binding kinetics taking into consideration data from Mn coordination complexes. This analysis supports a model in which the substrate waters are both bound as terminal ligands and react via a water-nucleophile attack mechanism.

  19. Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

    Science.gov (United States)

    Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

    2015-05-12

    VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

  20. Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

    Science.gov (United States)

    Rice, Austin J; Harrison, Alistair; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

    2014-05-23

    Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Resonance Raman study on indoleamine 2,3-dioxygenase: Control of reactivity by substrate-binding

    Energy Technology Data Exchange (ETDEWEB)

    Yanagisawa, Sachiko; Hara, Masayuki [Graduate School of Life Science and Picobiology Institute, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); Sugimoto, Hiroshi; Shiro, Yoshitsugu [Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Koto 1-1-1, Sayo-cho, Sayo-gun, Hyogo 679-5198 (Japan); Ogura, Takashi, E-mail: ogura@sci.u-hyogo.ac.jp [Graduate School of Life Science and Picobiology Institute, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan)

    2013-06-20

    Highlights: • Indoleamine 2,3-dioygenase has been studied by resonance Raman spectroscopy. • Trp-binding to the enzyme induces high frequency shift of the Fe–His stretching mode. • Increased imidazolate character of histidine promotes the O–O bond cleavage step. • A fine-tuning of the reactivity of the O–O bond cleavage reaction is identified. • The results are consistent with the sequential oxygen-atom-transfer mechanism. - Abstract: Resonance Raman spectra of ligand-bound complexes including the 4-phenylimidazole complex and of free and L-Trp-bound forms of indoleamine 2, 3-dioxygenase in the ferric state were examined. Effects on the vinyl and propionate substituent groups of the heme were detected in a ligand-dependent fashion. The effects of phenyl group of 4-phenylimidazole on the vinyl and propionate Raman bands were evident when compared with the case of imidazole ligand. Substrate binding to the ferrous protein caused an upshift of the iron–histidine stretching mode by 3 cm{sup −1}, indicating an increase in negativity of the imidazole ring, which favors the O–O bond cleavage. The substrate binding event is likely to be communicated from the heme distal side to the iron–histidine bond through heme substituent groups and the hydrogen-bond network which includes water molecules, as identified in an X-ray structure of a 4-phenylimidazole complex. The results provide evidence for fine-tuning of the reactivity of O–O bond cleavage by the oxygenated heme upon binding of L-Trp.

  2. The tight binding model study of the role of band filling on the charge gap in graphene-on-substrate in paramagnetic state

    Science.gov (United States)

    Panda, Rudrashish; Sahu, Sivabrata; Rout, G. C.

    2017-05-01

    We communicate here a tight binding theoretical model study of the band filling effect on the charge gap in graphene-on-substrate. The Hamiltonian consists of nearest neighbor electron hopping and substrate induced gap. Besides this the Coulomb interaction is considered here within mean-field approximation in the paramagnetic limit. The electron occupancies at two sublattices are calculated by Green's function technique and are solved self consistently. Finally the charge gap i.e. Δ ¯=U [ - ] is calculated and computed numerically. The results are reported.

  3. Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase

    DEFF Research Database (Denmark)

    Kramhøft, Birte; Bak-Jensen, Kristian Sass; Mori, Haruhide

    2005-01-01

    Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1...... translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products...

  4. Manipulating molecule-substrate exchange interactions via graphene

    Science.gov (United States)

    Bhandary, Sumanta; Eriksson, Olle; Sanyal, Biplab

    2013-03-01

    Organometallic molecules with a 3d metal center carrying a spin offers many interesting properties, e.g., existence of multiple spin states. A recent interest has been in understanding the magnetic exchange interaction between these organometallic molecules and magnetic substrates both from experiments and theory. In this work, we will show by calculations based on density functional theory how the exchange interaction is mediated via graphene in a geometry containing iron porphyrin(FeP)/graphene/Ni(111). The exchange interaction varies from a ferromagnetic to an antiferromagnetic one depending on the lattice site and type of defect in the graphene lattice along with the switching of spin state of Fe in FeP between S=1 and S=2, which should be detectable by x-ray magnetic circular dichroism experiments. This scenario of complex magnetic couplings with large magnetic moments may offer a unique spintronic logic device. We acknowledge financial support from the Swedish Research Council, KAW foundation and the ERC(project 247062 - ASD).

  5. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

    Science.gov (United States)

    Polte, T R; Hanks, S K

    1995-11-07

    The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

  6. Binding and thermodynamics of REV peptide-ctDNA interaction.

    Science.gov (United States)

    Upadhyay, Santosh Kumar

    2017-03-01

    The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a T m of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the T m by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive B→Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔC p ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically

  7. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    Science.gov (United States)

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

  8. Real-time sensing of epithelial cell-cell and cell-substrate interactions by impedance spectroscopy on porous substrates

    Energy Technology Data Exchange (ETDEWEB)

    Mondal, D.; RoyChaudhuri, C., E-mail: chirosreepram@yahoo.com [Department of Electronics and Telecommunication Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711103 (India); Pal, D. [Department of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711103 (India)

    2015-07-28

    Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion, we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (R{sub b}) corresponding to the initial adhesion phase of cells. It is observed that R{sub b} and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions.

  9. Real-time sensing of epithelial cell-cell and cell-substrate interactions by impedance spectroscopy on porous substrates

    International Nuclear Information System (INIS)

    Mondal, D.; RoyChaudhuri, C.; Pal, D.

    2015-01-01

    Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion, we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (R b ) corresponding to the initial adhesion phase of cells. It is observed that R b and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions

  10. Binding of MCM-interacting proteins to ATP-binding site in MCM6

    Directory of Open Access Journals (Sweden)

    Hosoi A

    2016-03-01

    Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

  11. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  12. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    Science.gov (United States)

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

  13. Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

    Science.gov (United States)

    Kin, Ng Hong; Ling, Tan Aik

    2016-01-01

    The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

  14. The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance linked ATP binding cassette drug transporter ABCG2.

    Science.gov (United States)

    Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V

    2007-12-01

    Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2, which are essential for blood clotting. The naturally occurring structural analogue of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We here report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). Vitamin K3 and plumbagin inhibited the binding of [(125)I]iodoarylazidoprazosin, a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC(50) values of 7.3 and 22.6 micromol/L, respectively, but had no effect on the binding of the photoaffinity analogue to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of the ABCG2 transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared with the control cells, suggesting that they are substrates of this transporter. Collectively, these data show for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function.

  15. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

    Science.gov (United States)

    Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

    1993-06-25

    Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

  16. Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

    Science.gov (United States)

    Swedberg, Joakim E; Harris, Jonathan M

    2011-10-04

    Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

  17. Molecular dynamics investigations of regioselectivity of anionic/aromatic substrates by a family of enzymes: a case study of diclofenac binding in CYP2C isoforms.

    Science.gov (United States)

    Cui, Ying-Lu; Xu, Fang; Wu, Rongling

    2016-06-29

    The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided.

  18. Interactions of barley alpha-amylase isozymes with Ca2+, substrates and proteinaceous inhibitors

    DEFF Research Database (Denmark)

    Abou Hachem, Maher; Bozonnet, Sophie; Willemoes, Martin

    2006-01-01

    discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other...

  19. Lack of interaction between the peptidomimetic substrates captopril and cephradine.

    Science.gov (United States)

    Foster, David R; Yee, Shiyin; Bleske, Barry E; Carver, Peggy L; Shea, Michael J; Menon, Sujatha S; Ramachandran, Chandrasekharan; Welage, Lynda S; Amidon, Gordon L

    2009-03-01

    Intestinal peptide transporters, including hPEPT1, facilitate the absorption of cephalosporins and angiotensin-converting enzyme inhibitors, and have been investigated as a means to improve oral drug absorption. Renal peptide transporters including hPEPT2, may also facilitate renal reabsorption of such compounds. In vitro and animal studies suggest that co-administration of peptidomimetic compounds may alter oral pharmacokinetics, although this has not been well studied in humans. The purpose of this study was to determine whether co-administration of the hPEPT substrates captopril and cephradine alters the oral pharmacokinetics of either agent. Nine healthy male volunteers received a single oral 25-mg dose of captopril, a single oral 500-mg dose of cephradine, or concurrent ingestion of captopril and cephradine in a cross-over manner. Venous blood samples were taken and captopril and cephradine pharmacokinetics were determined using noncompartmental analyses. No significant differences were observed in captopril or cephradine pharmacokinetics when administered together as compared to each agent alone (a marginal decrease in C(max) was observed for both captopril and cephradine during co-administration [5-15%]; however, differences were not statistically significant). The results of our study suggest that hPEPT1 and hPEPT2 are unlikely to contribute to clinically important drug interactions in humans.

  20. DNA-assisted binding of microspheres on glass substrates and their laser-induced release

    International Nuclear Information System (INIS)

    Kim, Sejong; Geiss, Erik; Yu, Phillip; Papadimitrakopoulos, Fotios; Marcus, Harris L.

    2006-01-01

    DNA hybridization has been increasingly adopted in materials sciences due to its complementary nature of single stranded DNAs. This unique property could be potentially used in the realization of 2 dimensional (2D) arrays of colloidal microspheres as a precursor to further build more complicated superstructures. In order to precisely understand this DNA-assisted assembly of colloidal particles, we quantitatively assessed the surface density of grafted and hybridizing accessible DNA oligomers on both substrate and colloidal particles. The DNA grafting densities were determined by UV-Vis of dye-functionalized complementary DNA oligomers, in conjunction with theoretical models. The variations of the concentration of hybridized DNA as a function of parameters such as the number of DNA base pairs (bp), the length of spacer and the size of sphere were also investigated to determine the immobilization strength of colloidal microspheres on the substrate. Dehybridization of the particle was conducted by utilizing a focused laser beam. These results were also compared with the particle hybridization energies and modeled according to the sum of DNA bindings as a function of the number of hybridized bases

  1. Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao; Xue, Fei; Wu, Zhixin; Shen, Wei; Li, Jun; Zhou, Zhe; Ding, Yi; Zhao, Qi; Zhang, Xuejun C.; Liao, Ming; Bartlam, Mark; Rao, Zihe (SCAU); (Tsinghua); (Chinese Aca. Sci.)

    2008-07-21

    Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) MP{sup pro} and a severe acute respiratory syndrome CoV (SARS-CoV) M{sup pro} mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M{sup pro}. A monomeric form of IBV M{sup pro} was identified for the first time in CoV M{sup pro} structures. A comparison of these two structures to other available M{sup pro} structures provides new insights for the design of substrate-based inhibitors targeting CoV M{sup pro}s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M{sup pro} and was found to demonstrate in vitro inactivation of IBV M{sup pro} and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M{sup pro}.

  2. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    Science.gov (United States)

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  3. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding.

    Science.gov (United States)

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

  4. NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions.

    Science.gov (United States)

    Harris, Kimberly A; Bobay, Benjamin G; Sarachan, Kathryn L; Sims, Alexis F; Bilbille, Yann; Deutsch, Christopher; Iwata-Reuyl, Dirk; Agris, Paul F

    2015-08-14

    The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

    International Nuclear Information System (INIS)

    Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

    1991-01-01

    Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

  6. Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD.

    Science.gov (United States)

    Mireku, S A; Sauer, M M; Glockshuber, R; Locher, K P

    2017-10-30

    Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10 -6 and 10 -9  M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 10 4 and 10 6  M -1 s -1 . For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.

  7. STRUCTURAL INSIGHTS INTO SUBSTRATE BINDING AND STEREOSELECTIVITY OF GIARDIA FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE*

    Science.gov (United States)

    Galkin, Andrey; Li, Zhimin; Li, Ling; Kulakova, Liudmila; Pal, Lipika R.; Dunaway-Mariano, Debra; Herzberg, Osnat

    2009-01-01

    Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA)1 is a member of the Class II zinc-dependent aldolase family that catalyzes the cleavage of D-fructose-1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced by an alanine residue renders the enzyme inactive. A comparison of the crystal structures of the D83A FBPA in complex with FBP and of the wild-type FBPA in the unbound state revealed a substrate induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn2+. Upon FBP binding, the Zn2+ shifts up to 4.6 Å towards the catalytic Asp83, which brings the metal within coordination distance to the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor D-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analog of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA/FBP structure. The mechanism invokes coordination and co-planarity of the Zn2+ with the FBP’s O-C(3)-C(4)-O concomitant with coordination of Asp83 carboxylic group. Catalysis is accompanied by movement of Zn2+ to a site co-planar with the O-C(2)-C(3)-O of the DHAP. glFBPA exhibit strict substrate specificity towards FBP and

  8. Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

    Science.gov (United States)

    Chen, Kai; Duan, Wenxiu; Han, Qianqian; Sun, Xuan; Li, Wenqian; Hu, Shuangyun; Wan, Jiajia; Wu, Jiang; Ge, Yushu; Liu, Dan

    2018-03-08

    Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

  9. Adhesive interactions of geckos with wet and dry fluoropolymer substrates.

    Science.gov (United States)

    Stark, Alyssa Y; Dryden, Daniel M; Olderman, Jeffrey; Peterson, Kelly A; Niewiarowski, Peter H; French, Roger H; Dhinojwala, Ali

    2015-07-06

    Fluorinated substrates like Teflon® (poly(tetrafluoroethylene); PTFE) are well known for their role in creating non-stick surfaces. We showed previously that even geckos, which can stick to most surfaces under a wide variety of conditions, slip on PTFE. Surprisingly, however, geckos can stick reasonably well to PTFE if it is wet. In an effort to explain this effect, we have turned our attention to the role of substrate surface energy and roughness when shear adhesion occurs in media other than air. In this study, we removed the roughness component inherent to commercially available PTFE and tested geckos on relatively smooth wet and dry fluoropolymer substrates. We found that roughness had very little effect on shear adhesion in air or in water and that the level of fluorination was most important for shear adhesion, particularly in air. Surface energy calculations of the two fluorinated substrates and one control substrate using the Tabor-Winterton approximation and the Young-Dupré equation were used to determine the interfacial energy of the substrates. Using these interfacial energies we estimated the ratio of wet and dry normal adhesion for geckos clinging to the three substrates. Consistent with the results for rough PTFE, our predictions show a qualitative trend in shear adhesion based on fluorination, and the quantitative experimental differences highlight the unusually low shear adhesion of geckos on dry smooth fluorinated substrates, which is not captured by surface energy calculations. Our work has implications for bioinspired design of synthetics that can preferentially stick in water but not in air.

  10. Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

    DEFF Research Database (Denmark)

    Erlendsson, Simon; Gotfryd, Kamil; Larsen, Flemming Hofmann

    2017-01-01

    The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been...

  11. Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding

    DEFF Research Database (Denmark)

    Fredslund, Folmer; Abou Hachem, Maher; Larsen, Rene Jonsgaard

    2011-01-01

    structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N...

  12. A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

    Science.gov (United States)

    Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

    2010-12-01

    Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

  13. A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data*

    Science.gov (United States)

    Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

    2010-01-01

    Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/. PMID:20733106

  14. Titanium diboride coatings and their interaction with the substrates

    International Nuclear Information System (INIS)

    Pierson, H.O.; Randich, E.

    1978-01-01

    An experimental investigation of the chemical vapor deposition (CVD) of titanium diboride (TiB 2 ) on metallic substrates, using the hydrogen reduction of TiCl 4 and BCl 3 at 1 atmosphere and at temperatures between 850 0 C and 1050 0 C is described. To be coated, the substrate had to meet the following requirements: (1) ability to withstand the deposition temperature without detrimental transformation, (2) chemical inertness to the by-products of the reaction (mostly HCl), (3) reasonable matching of its thermal expansion with that of TiB 2 . The latter requirement may be partially circumvented by using a ductile intermediate coating such as Cu or Ni. Substrates meeting these requirements were W, Ta, Ni, WC, TiC, Kovar and some high chrome steels. Coatings on these substrates were examined by metallographic techniques, scanning electron microscope, x-ray diffraction and electron microprobe. The structures and the degree of interdiffusion were determined. In most cases, intermediate borides of the type M 3 B and M 2 B were formed. The hardness of the coatings was 3330 +- 310 kg/mm 2 (VHN 50 ). Coatings of TiB 2 have already been used successfully on letdown valves in a bench scale coal liquefaction reactor at Sandia Laboratories

  15. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  16. Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

    Science.gov (United States)

    Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2014-03-18

    Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Structure formation in bis(terpyridine) derivative adlayers: molecule-substrate versus molecule-molecule interactions.

    Science.gov (United States)

    Hoster, Harry E; Roos, Matthias; Breitruck, Achim; Meier, Christoph; Tonigold, Katrin; Waldmann, Thomas; Ziener, Ulrich; Landfester, Katharina; Behm, R Jürgen

    2007-11-06

    The influence of the substrate and the deposition conditions-vapor deposition versus deposition from solution-on the structures formed upon self-assembly of deposited bis(terpyridine) derivative (2,4'-BTP) monolayers on different hexagonal substrates, including highly oriented pyrolytic graphite (HOPG), Au(111), and (111)-oriented Ag thin films, was investigated by high-resolution scanning tunneling microscopy and by model calculations of the intermolecular energies and the lateral corrugation of the substrate-adsorbate interaction. Similar quasi-quadratic network structures with almost the same lattice constants obtained on all substrates are essentially identical to the optimum configuration expected from an optimization of the adlayer structure with C-H...N-type bridging bonds as a structure-determining factor, which underlines a key role of the intermolecular interactions in adlayer order. Slight distortions from the optimum values to form commensurate adlayer structures on the metal substrates and the preferential orientation of the adlayer with respect to the substrate are attributed to the substrate-adsorbate interactions, specifically, the lateral corrugation in the substrate-adsorbate interaction upon lateral displacement and rotation of the adsorbed BTP molecules. The fact that similar adlayer structures are obtained on HOPG under ultrahigh vacuum conditions (solid|gas interface) and on HOPG in trichlorobenzene (solid|liquid interface) indicates that the intermolecular interactions are not severely affected by the solvent.

  18. Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Elferich, Johannes; Gouaux, Eric (Oregon HSU)

    2012-02-13

    Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-{beta}-D-glucopyranoside ({beta}-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT-substrate complexes in lipid bicelles and in {beta}-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

  19. Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Bak-Jensen, K.S.; André, G.; Gottschalk, T.E.

    2004-01-01

    and oligosaccharides, respectively. Bond cleavage analysis of oligosaccharide degradation by wild-type and mutant AMY1 supports that Tyr105 is critical for binding at subsite -6. Substrate binding is improved by T212(Y/W) introduced at subsite +4 and the [Y105A/ T212(Y/W)] AMY1 double mutants synergistically enhanced......The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites - 6 and +4 ( substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved...... in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and 1% activity (k(cat)/K-m) on starch, amylose DP17, and 2-chloro-4-nitrophenyl β-D-maltoheptaoside, whereas T212Y at subsite +4 has 32, 370, and 90...

  20. Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping.

    Directory of Open Access Journals (Sweden)

    Avraham Ben-Shimon

    2011-11-01

    Full Text Available Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P-2 and P-5, commonly occupied by Arginine (Arg in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P-2/P-5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P-5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering. The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P-2 and P-5 positions.

  1. Substrate-HTcS thin film interaction studies by (S)TEM

    NARCIS (Netherlands)

    Ramaekers, P.P.J.; Klepper, D.; Kitazawa, K.; Ishiguro, T.

    1989-01-01

    This paper concerns with compatibility aspects beween HTcS thin film either their substrates. The influence of substrate-thin film interaction and thin film microstructure on the superconducting properties is discussed. In this respect, data based on (S)TEM observations are presented. It is

  2. Reconstitution of high affinity α2 adrenergic agonist binding by fusion with a pertussis toxin substrate

    International Nuclear Information System (INIS)

    Kim, M.H.; Neubig, R.R.

    1986-01-01

    High affinity α 2 adrenergic agonist binding is thought to occur via a coupling of the α 2 receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 μM phenoxybenzamine to block α 2 receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the α 2 agonist [ 3 H]UK 14,304 (UK) and the antagonist [ 3 H] yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain α 2 receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance [ 3 H] UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity α 2 agonist binding

  3. Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study

    DEFF Research Database (Denmark)

    da Graça Thrige, D; Buur, J R; Jørgensen, Flemming Steen

    1997-01-01

    cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between...... water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations...... the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular...

  4. Effects of multiple enzyme–substrate interactions in basic units of cellular signal processing

    International Nuclear Information System (INIS)

    Seaton, D D; Krishnan, J

    2012-01-01

    Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme–substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme–substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein–protein interactions are important in determining the signalling properties of enzymatic signalling pathways. (paper)

  5. Biochemistry students' ideas about shape and charge in enzyme-substrate interactions.

    Science.gov (United States)

    Linenberger, Kimberly J; Bretz, Stacey Lowery

    2014-01-01

    Biochemistry is a visual discipline that requires students to develop an understanding of numerous representations. However, there is very little known about what students actually understand about the representations that are used to communicate ideas in biochemistry. This study investigated biochemistry students' understanding of multiple representations of enzyme-substrate interactions through both student interviews (N = 25) and responses by a national sample (N = 707) to the Enzyme-Substrate Interactions Concept Inventory. This manuscript reports the findings regarding one category of misconceptions measured by the concept inventory, namely, students' understandings of shape and charge in the context of enzyme-substrate interactions. Students interpret molecular representations depicting such interactions by determining the complementarity between enzyme and substrate by focusing upon charge and hydrogen bonding, but with a disregard for stereochemistry. Copyright © 2014 by The International Union of Biochemistry and Molecular Biology.

  6. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across...... the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes...

  7. Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

    Science.gov (United States)

    Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

    2016-03-14

    Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

  8. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation.

    Directory of Open Access Journals (Sweden)

    Luigi Capoferri

    Full Text Available Prediction of human Cytochrome P450 (CYP binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD simulations and Linear Interaction Energy (LIE theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE of 4.1 kJ mol-1 and a standard error in prediction (SDEP in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units.

  9. A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

    DEFF Research Database (Denmark)

    Wong, W T; Schumacher, C; Salcini, A E

    1995-01-01

    In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins....

  10. Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

    Directory of Open Access Journals (Sweden)

    Stephanie J. Ruiz

    2016-12-01

    Full Text Available BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related compounds have failed to demonstrate transport mediated by BilE. The putative substrate-binding domain (SBD of BilE was expressed in isolation and the crystal structure solved at 1.5 Å. Although the overall fold is characteristic of SBDs, the binding site varies considerably relative to the well-characterized homologs ProX from Archaeoglobus fulgidus and OpuBC and OpuCC from Bacillus subtilis. This suggests that BilE may bind an as-yet unknown ligand. Elucidation of the natural substrate of BilE could reveal a novel bile resistance mechanism.

  11. Osteoblast interaction with DLC-coated Si substrates.

    Science.gov (United States)

    Chai, Feng; Mathis, Nicolas; Blanchemain, Nicolas; Meunier, Cathy; Hildebrand, Hartmut F

    2008-09-01

    Diamond-like carbon (DLC) coating is a convenient means of modifying material surfaces that are sensitive to wear, such as titanium and silica substrates. This work aims to evaluate the osteoblast-like cells' response to DLC-coated Si (Si-DLC), which was treated under different conditions. DLC and deuterated DLC films were deposited by plasma-enhanced chemical vapor deposition to obtain a 200-nm-thick layer on all the samples. Three types of precursor gas were applied for deposition: pure methane (CH(4)), pure deuterated methane (CD(4)) and their half/half mixture. All surface treatments were performed under two different self-bias voltages (V(sb)): -400 and -600V. The modified surfaces were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, Rutherford backscattering spectroscopy, elastic recoil detection analysis, X-ray reflectometry and the sessile-drop method. MC3T3-E1 osteoblasts were cultured on the Si-DLC wafers for 3 and 6 days. Biological tests to measure cell proliferation, cell vitality, cell morphology and cell adhesion were performed. All DLC coatings produced a slightly more hydrophobic state than non-treated Si. Certain types of amorphous DLC coating, such as the surface treated under the V(sb) of -600V in pure methane (600CH(4)) or in pure deuterated methane (600CD(4)), offered a significantly higher cell proliferation rate to Si substrate. Scanning electron microscopy observations confirmed that the optimal cell adhesion behavior, among all the treated surfaces, occurred on the surface of the 600CH(4) and 600CD(4) groups, which showed increased amounts of filopodia and microvilli to enhance cell-environment exchange. In conclusion, DLC coating on Si could produce better surface stability and improved cellular responses.

  12. Substrate Binding Drives Active-Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot-Spot Labeling and NMR Spectroscopy Experiments.

    Science.gov (United States)

    Weissbach, Sophie; Flügge, Friedemann; Peters, Thomas

    2018-05-04

    Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized 15 N side-chain labeling of Trp residues and 13 C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Mechanism-based population pharmacokinetic modelling in diabetes: vildagliptin as a tight binding inhibitor and substrate of dipeptidyl peptidase IV

    Science.gov (United States)

    Landersdorfer, Cornelia B; He, Yan-Ling; Jusko, William J

    2012-01-01

    AIMS To assess the pharmacokinetics of vildagliptin at different doses and build a mechanism-based population model that simultaneously describes vildagliptin pharmacokinetics and its effects on DPP-4 activity based on underlying physiology and biology. METHODS Vildagliptin concentrations and DPP-4 activity vs. time from 13 type 2 diabetic patients after oral vildagliptin 10, 25 or 100 mg and placebo twice daily for 28 days were co-modelled. NONMEM VI and S-ADAPT were utilized for population modelling. RESULTS A target-mediated drug disposition (TMDD) model accounting for capacity-limited high affinity binding of vildagliptin to DPP-4 in plasma and tissues had good predictive performance. Modelling the full time course of the vildagliptin-DPP-4 interaction suggested parallel vildagliptin dissociation from DPP-4 by a slow first-order process and hydrolysis by DPP-4 to an inactive metabolite as a disposition mechanism. Due to limited amounts of DPP-4, vildagliptin concentrations increased slightly more than dose proportionally. This newly proposed model and the parameter estimates are supported by published in vitro studies. Mean parameter estimates (inter-individual coefficient of variation) were: non-saturable clearance 36 l h−1 (25%), central volume of distribution 22 l (37%), half-life of dissociation from DPP-4 1.1 h (94%) and half-life of hydrolysis 6.3 h (81%). CONCLUSIONS Vildagliptin is both an inhibitor and substrate for DPP-4. By utilizing the TMDD approach, slow dissociation of vildagliptin from DPP-4 was found in patients and the half-life of hydrolysis by DPP-4 estimated. This model can be used to predict DPP-4 inhibition effects of other dosage regimens and be modified for other DPP-4 inhibitors to differentiate their properties. PMID:22442826

  14. Spectroscopic investigation of new water soluble Mn(II)(2) and Mg(II)(2) complexes for the substrate binding models of xylose/glucose isomerases.

    Science.gov (United States)

    Patra, Ayan; Bera, Manindranath

    2014-01-30

    In methanol, the reaction of stoichiometric amounts of Mn(OAc)(2)·4H(2)O and the ligand H(3)hpnbpda [H(3)hpnbpda=N,N'-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N'-diacetic acid] in the presence of NaOH, afforded a new water soluble dinuclear manganese(II) complex, [Mn2(hpnbpda)(μ-OAc)] (1). Similarly, the reaction of Mg(OAc)(2)·4H(2)O and the ligand H3hpnbpda in the presence of NaOH, in methanol, yielded a new water soluble dinuclear magnesium(II) complex, [Mg2(hpnbpda)(μ-OAc)(H2O)2] (2). DFT calculations have been performed for the structural optimization of complexes 1 and 2. The DFT optimized structure of complex 1 shows that two manganese(II) centers are in a distorted square pyramidal geometry, whereas the DFT optimized structure of complex 2 reveals that two magnesium(II) centers adopt a six-coordinate distorted octahedral geometry. To understand the mode of substrate binding and the mechanistic details of the active site metals in xylose/glucose isomerases (XGI), we have investigated the binding interactions of biologically important monosaccharides d-glucose and d-xylose with complexes 1 and 2, in aqueous alkaline solution by a combined approach of FTIR, UV-vis, fluorescence, and (13)C NMR spectroscopic techniques. Fluorescence spectra show the binding-induced gradual decrease in emission of complexes 1 and 2 accompanied by a significant blue shift upon increasing the concentration of sugar substrates. The binding modes of d-glucose and d-xylose with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in (13)C NMR spectra for C1 and C2 carbon atoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Insights into phosphate cooperativity and influence of substrate modifications on binding and catalysis of hexameric purine nucleoside phosphorylases.

    Directory of Open Access Journals (Sweden)

    Priscila O de Giuseppe

    Full Text Available The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233 displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxyribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43* side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl(6 and Br(8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91 is responsible for the lack of negative cooperativity of phosphate binding

  16. Effect of Peptides' Binding on the Antimicrobial Activity and Biocompatibility of Protein-Based Substrates

    Science.gov (United States)

    Kaisersberger Vincek, Maja

    This work reveals the effect of coupling approach (chemical by using carbodiimide chemistry and grafting-to vs. grafting-from synthesis routes, and enzymatic by using transglutaminase) of a hydrophilic epsilon-poly-L-lysine (epsilonPL) and an amphiphilic oligo-acyl-lysyl (OAK) derivative (K-7alpha 12-OH) to wool fibers and gelatine (GEL) macromolecules, respectively, and substrates antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus bacteria after 1-24 h of exposure, as well as their cytotoxicity. Different spectroscopic (ultraviolet-visible, infrared, fluorescence and electron paramagnetic resonance) and separation techniques (size-exclusion chromatography and capillary zone electrophoresis) as well as zeta potential and potentiometric titration analysis, were performed to confirm the covalent coupling of epsilonPL/OAK, and to determine the amount and orientation of its immobilisation. The highest and kinetically the fastest level of bacterial reduction was achieved with wool/GEL functionalised with epsilonPL/OAK by chemical grafting-to approach. This effect correlated with both the highest grafting yield and conformationally the highly-flexible (brush-like) orientation linkage of epsilonPL/OAK, implicating on the highest amount of accessible amino groups interacting with bacterial membrane. However, OAK's amphipathic structure, the cationic charge and the hydrophobic moieties, resulted to relatively high reduction of S. aureus for grafting-from and the enzymatic coupling approaches using OAK-functionalised GEL. The epsilonPL/OAK-functionalised GEL did not induce toxicity in human osteoblast cells, even at 25-fold higher concentration than bacterial minimum inhibitory (MIC) concentration of epsilonPL/OAK, supporting their potential usage in biomedical applications. It was also shown that non-ionic surfactant adsorbs strongly onto the wool surface during the process of washing, thereby blocking the functional sites of immobilized

  17. Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach.

    Science.gov (United States)

    Opalińska, Magdalena; Parys, Katarzyna; Jańska, Hanna

    2017-11-18

    Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i -AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4's in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4's physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

  18. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  19. Integration of statistical modeling and high-content microscopy to systematically investigate cell-substrate interactions.

    Science.gov (United States)

    Chen, Wen Li Kelly; Likhitpanichkul, Morakot; Ho, Anthony; Simmons, Craig A

    2010-03-01

    Cell-substrate interactions are multifaceted, involving the integration of various physical and biochemical signals. The interactions among these microenvironmental factors cannot be facilely elucidated and quantified by conventional experimentation, and necessitate multifactorial strategies. Here we describe an approach that integrates statistical design and analysis of experiments with automated microscopy to systematically investigate the combinatorial effects of substrate-derived stimuli (substrate stiffness and matrix protein concentration) on mesenchymal stem cell (MSC) spreading, proliferation and osteogenic differentiation. C3H10T1/2 cells were grown on type I collagen- or fibronectin-coated polyacrylamide hydrogels with tunable mechanical properties. Experimental conditions, which were defined according to central composite design, consisted of specific permutations of substrate stiffness (3-144 kPa) and adhesion protein concentration (7-520 microg/mL). Spreading area, BrdU incorporation and Runx2 nuclear translocation were quantified using high-content microscopy and modeled as mathematical functions of substrate stiffness and protein concentration. The resulting response surfaces revealed distinct patterns of protein-specific, substrate stiffness-dependent modulation of MSC proliferation and differentiation, demonstrating the advantage of statistical modeling in the detection and description of higher-order cellular responses. In a broader context, this approach can be adapted to study other types of cell-material interactions and can facilitate the efficient screening and optimization of substrate properties for applications involving cell-material interfaces. Copyright 2009 Elsevier Ltd. All rights reserved.

  20. Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.

    Science.gov (United States)

    Wei, Pengcheng; Song, Guangji; Shi, Mengyang; Zhou, Yafei; Liu, Yang; Lei, Jun; Chen, Peng; Yin, Lei

    2018-02-01

    Colistin is considered a last-resort antibiotic against most gram-negative bacteria. Recent discoveries of a plasmid-mediated, transferable mobilized colistin-resistance gene ( mcr-1) on all continents have heralded the imminent emergence of pan-drug-resistant superbacteria. The inner-membrane protein MCR-1 can catalyze the transfer of phosphoethanolamine (PEA) to lipid A, resulting in colistin resistance. However, little is known about the mechanism, and few drugs exist to address this issue. We present crystal structures revealing the MCR-1 catalytic domain (cMCR-1) as a monozinc metalloprotein with ethanolamine (ETA) and d-glucose, respectively, thus highlighting 2 possible substrate-binding pockets in the MCR-1-catalyzed PEA transfer reaction. Mutation of the residues involved in ETA and d-glucose binding impairs colistin resistance in recombinant Escherichia coli containing full-length MCR-1. Partial analogs of the substrate are used for cocrystallization with cMCR-1, providing valuable information about the family of PEA transferases. One of the analogs, ETA, causes clear inhibition of polymyxin B resistance, highlighting its potential for drug development. These data demonstrate the crucial role of the PEA- and lipid A-binding pockets and provide novel insights into the structure-based mechanisms, important drug-target hot spots, and a drug template for further drug development to combat the urgent, rising threat of MCR-1-mediated antibiotic resistance.-Wei, P., Song, G., Shi, M., Zhou, Y., Liu, Y., Lei, J., Chen, P., Yin, L. Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.

  1. Visualising substrate-fingermark interactions: Solid-state NMR spectroscopy of amino acid reagent development on cellulose substrates.

    Science.gov (United States)

    Spindler, Xanthe; Shimmon, Ronald; Roux, Claude; Lennard, Chris

    2015-05-01

    Most spectroscopic studies of the reaction products formed by ninhydrin, 1,2-indanedione-zinc (Ind-Zn) and 1,8-diazafluoren-9-one (DFO) when reacted with amino acids or latent fingermarks on paper substrates are focused on visible absorption or luminescence spectroscopy. In addition, structural elucidation studies are typically limited to solution-based mass spectrometry or liquid nuclear magnetic resonance (NMR) spectroscopy, which does not provide an accurate representation of the fingermark development process on common paper substrates. The research presented in this article demonstrates that solid-state carbon-13 magic angle spinning NMR ((13)C-MAS-NMR) is a technique that can not only be utilised for structural studies of fingermark enhancement reagents, but is a promising technique for characterising the effect of paper chemistry on fingermark deposition and enhancement. The latter opens up a research area that has been under-explored to date but has the potential to improve our understanding of how fingermark secretions and enhancement reagents interact with paper substrates. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. ER-bound protein tyrosine phosphatase PTP1B interacts with Src at the plasma membrane/substrate interface.

    Directory of Open Access Journals (Sweden)

    Melisa C Monteleone

    Full Text Available PTP1B is an endoplasmic reticulum (ER anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC. Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

  3. TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity.

    Science.gov (United States)

    Pereira, L A; van der Knaap, J A; van den Boom, V; van den Heuvel, F A; Timmers, H T

    2001-11-01

    The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

  4. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.

    Science.gov (United States)

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

    2015-06-05

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

    Science.gov (United States)

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

    2015-01-01

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

  6. Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy.

    Science.gov (United States)

    Loewen, Peter C; Villanueva, Jacylyn; Switala, Jacek; Donald, Lynda J; Ivancich, Anabella

    2015-05-01

    Heme-containing catalases and catalase-peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase-peroxidase led us to investigate the enzyme for comparison with other catalase-peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat  = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat  = 20 s(-1) ) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65-1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the Fe(III) high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. © 2015 Wiley Periodicals, Inc.

  7. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    Science.gov (United States)

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  8. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

    Directory of Open Access Journals (Sweden)

    Mykuliak V. V.

    2014-03-01

    Full Text Available Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS. Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

  9. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    Science.gov (United States)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  10. Interaction of Object Binding Cues in Binaural Masking Pattern Experiments.

    Science.gov (United States)

    Verhey, Jesko L; Lübken, Björn; van de Par, Steven

    2016-01-01

    Object binding cues such as binaural and across-frequency modulation cues are likely to be used by the auditory system to separate sounds from different sources in complex auditory scenes. The present study investigates the interaction of these cues in a binaural masking pattern paradigm where a sinusoidal target is masked by a narrowband noise. It was hypothesised that beating between signal and masker may contribute to signal detection when signal and masker do not spectrally overlap but that this cue could not be used in combination with interaural cues. To test this hypothesis an additional sinusoidal interferer was added to the noise masker with a lower frequency than the noise whereas the target had a higher frequency than the noise. Thresholds increase when the interferer is added. This effect is largest when the spectral interferer-masker and masker-target distances are equal. The result supports the hypothesis that modulation cues contribute to signal detection in the classical masking paradigm and that these are analysed with modulation bandpass filters. A monaural model including an across-frequency modulation process is presented that account for this effect. Interestingly, the interferer also affects dichotic thresholds indicating that modulation cues also play a role in binaural processing.

  11. Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative*

    OpenAIRE

    Kashiwayama, Yoshinori; Tomohiro, Takenori; Narita, Kotomi; Suzumura, Miyuki; Glumoff, Tuomo; Hiltunen, J. Kalervo; Van Veldhoven, Paul P.; Hatanaka, Yasumaru; Imanaka, Tsuneo

    2010-01-01

    Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a d...

  12. Cell-substrate interaction with cell-membrane-stress dependent adhesion.

    Science.gov (United States)

    Jiang, H; Yang, B

    2012-01-10

    Cell-substrate interaction is examined in a two-dimensional mechanics model. The cell and substrate are treated as a shell and an elastic solid, respectively. Their interaction through adhesion is treated using nonlinear springs. Compared to previous cell mechanics models, the present model introduces a cohesive force law that is dependent not only on cell-substrate distance but also on internal cell-membrane stress. It is postulated that a living cell would establish focal adhesion sites with density dependent on the cell-membrane stress. The formulated mechanics problem is numerically solved using coupled finite elements and boundary elements for the cell and the substrate, respectively. The nodes in the adhesion zone from either side are linked by the cohesive springs. The specific cases of a cell adhering to a homogeneous substrate and a heterogeneous bimaterial substrate are examined. The analyses show that the substrate stiffness affects the adhesion behavior significantly and regulates the direction of cell adhesion, in good agreement with the experimental results in the literature. By introducing a reactive parameter (i.e., cell-membrane stress) linking biological responses of a living cell to a mechanical environment, the present model offers a unified mechanistic vehicle for characterization and prediction of living cell responses to various kinds of mechanical stimuli including local extracellular matrix and neighboring cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

    Directory of Open Access Journals (Sweden)

    Garima Tiwari

    Full Text Available Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i or K(d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i or K(d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors

  14. Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

    Science.gov (United States)

    Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

    2010-01-01

    Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

  15. Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

    Science.gov (United States)

    Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

    2017-06-06

    Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

  16. Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states

    DEFF Research Database (Denmark)

    Jensen, Kristine Steen; Winther, Jakob R; Teilum, Kaare

    2011-01-01

    to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions......Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover...... with a combination of NMR magnetization transfer, R(2) relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically...

  17. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    International Nuclear Information System (INIS)

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-01-01

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [ 32 P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-α/sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-α/sub i/ or anti-α 0 antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed

  18. Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

    Science.gov (United States)

    Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

    2013-08-01

    Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

  19. Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

    2003-08-30

    The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

  20. Functional group and stereochemical requirements for substrate binding by ghrelin O-acyltransferase revealed by unnatural amino acid incorporation.

    Science.gov (United States)

    Cleverdon, Elizabeth R; Davis, Tasha R; Hougland, James L

    2018-04-21

    Ghrelin is a small peptide hormone that undergoes a unique posttranslational modification, serine octanoylation, to play its physiological roles in processes including hunger signaling and glucose metabolism. Ghrelin O-acyltransferase (GOAT) catalyzes this posttranslational modification, which is essential for ghrelin to bind and activate its cognate GHS-R1a receptor. Inhibition of GOAT offers a potential avenue for modulating ghrelin signaling for therapeutic effect. Defining the molecular characteristics of ghrelin that lead to binding and recognition by GOAT will facilitate the development and optimization of GOAT inhibitors. We show that small peptide mimics of ghrelin substituted with 2,3-diaminopropanoic acid in place of the serine at the site of octanoylation act as submicromolar inhibitors of GOAT. Using these chemically modified analogs of desacyl ghrelin, we define key functional groups within the N-terminal sequence of ghrelin essential for binding to GOAT and determine GOAT's tolerance to backbone methylations and altered amino acid stereochemistry within ghrelin. Our study provides a structure-activity analysis of ghrelin binding to GOAT that expands upon activity-based investigations of ghrelin recognition and establishes a new class of potent substrate-mimetic GOAT inhibitors for further investigation and therapeutic interventions targeting ghrelin signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Dynamic fluorescence spectroscopy on single tryptophan mutants of EIImtl in detergent micelles : Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay

    NARCIS (Netherlands)

    Swaving Dijkstra, Dolf; Broos, J.; Visser, Antonie J.W.G.; van Hoek, A.; Robillard, George

    1997-01-01

    The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C

  2. Molecular dynamics investigation of nanoscale substrate topography and its interaction with liquids

    Science.gov (United States)

    Cordeiro Rodrigues, Jhonatam

    Nanotechnology has been presenting successful applications in several areas. However, experimentation with nanoscale materials is costly and limited in analysis capability. This research investigates the use of molecular dynamics (MD) simulations to model and study nanomaterials and manufacturing processes. MD simulations are employed to reduce cost, optimize design, increase productivity and allow for the investigation of material interactions not yet observable through experimentation. This work investigates the interaction of water with substrates at the nanoscale. The effect of temperature, droplet impingement velocities and size, as well as substrate material, are investigated at the nanoscale. Several substrate topography designs were modeled to reveal their influence on the wettability of the substrate. Nanoscale gold and silicon substrates are more hydrophilic at higher temperatures than at room temperature. The reduction in droplet diameter increases its wettability. High impingement velocity of droplets does not influence final wettability of substrates but induces higher diffusion rates of droplets in a heated environment. Droplets deposited over a gradient of surface exposure presents spontaneous movement. The Leidenfrost effect was investigated at the nanoscale. Droplets of 4 and 10nm in diameter presented behaviors pertinent to the Leidenfrost effect at 373K, significantly lower than at micro scale and of potential impact to the field. Topographical features were manipulated using superhydrophobic coating resulting in micro whiskers. Nanoimprint lithography (NIL) was used to manufacture substrate topographies at the nanoscale. Water droplets were deposited on the substrates and their wettability was measured using droplet contact angles. Lower surface area exposure resulted in higher contact angles. The experimental relationships between surface topography and substrate wettability were used to validate the insights gained from MD simulations for

  3. Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

    Science.gov (United States)

    Zgiby, S M; Thomson, G J; Qamar, S; Berry, A

    2000-03-01

    Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a

  4. A meta-selective C-H borylation directed by a secondary interaction between ligand and substrate

    Science.gov (United States)

    Kuninobu, Yoichiro; Ida, Haruka; Nishi, Mitsumi; Kanai, Motomu

    2015-09-01

    Regioselective C-H bond transformations are potentially the most efficient method for the synthesis of organic molecules. However, the presence of many C-H bonds in organic molecules and the high activation barrier for these reactions make these transformations difficult. Directing groups in the reaction substrate are often used to control regioselectivity, which has been especially successful for the ortho-selective functionalization of aromatic substrates. Here, we describe an iridium-catalysed meta-selective C-H borylation of aromatic compounds using a newly designed catalytic system. The bipyridine-derived ligand that binds iridium contains a pendant urea moiety. A secondary interaction between this urea and a hydrogen-bond acceptor in the substrate places the iridium in close proximity to the meta-C-H bond and thus controls the regioselectivity. 1H NMR studies and control experiments support the participation of hydrogen bonds in inducing regioselectivity. Reversible direction of the catalyst through hydrogen bonds is a versatile concept for regioselective C-H transformations.

  5. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.

    2006-01-01

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites...... as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile......, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in a-amylases....

  6. Effect of dispersion on surface interactions of cobalt(II) octaethylporphyrin monolayer on Au(111) and HOPG(0001) substrates: a comparative first principles study.

    Science.gov (United States)

    Chilukuri, Bhaskar; Mazur, Ursula; Hipps, K W

    2014-07-21

    A density functional theory study of a cobalt(II) octaethylporphyrin (CoOEP) monolayer on Au(111) and HOPG(0001) surfaces was performed under periodic boundary conditions. Calculations with and without dispersion corrections are performed and the effect of van der Waals forces on the interface properties is analyzed. Calculations have determined that the CoOEP molecule tends to bind at the 3-fold and the 6-fold center sites on Au(111) and HOPG(0001), respectively. Geometric optimizations at the center binding sites have indicated that the porphyrin molecules (in the monolayer) lie flat on both substrates. Calculations also reveal that the CoOEP monolayer binds slightly more strongly to Au(111) than to HOPG(0001). Charge density difference plots disclose that charge is redistributed mostly around the porphyrin plane and the first layer of the substrates. Dispersion interactions cause a larger substrate to molecule charge pushback on Au(111) than on HOPG. CoOEP adsorption tends to lower the work functions of either substrate, qualitatively agreeing with the experimental photoelectron spectroscopic data. Comparison of the density of states (DOS) of the isolated CoOEP molecule with that on gold and HOPG substrates showed significant band shifts around the Fermi energy due to intermolecular orbital hybridization. Simulated STM images were plotted with the Tersoff-Hamann approach using the local density of states, which also agree with the experimental results. This study elucidates the role of dispersion for better describing porphyrin-substrate interactions. A DFT based overview of geometric, adsorption and electronic properties of a porphyrin monolayer on conductive surfaces is presented.

  7. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  8. Substrate and cofactor binding to nitrile reductase : A mass spectrometry based study

    NARCIS (Netherlands)

    Gjonaj, L.; Pinkse, M.W.H.; Fernandez Fueyo, E.; Hollmann, F.; Hanefeld, U.

    2016-01-01

    Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman

  9. Substrate binding in the active site of cytochrome P450cam

    NARCIS (Netherlands)

    Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

    2005-01-01

    We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

  10. Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

    Science.gov (United States)

    Otero, Joel H; Lizák, Beata; Feige, Matthias J; Hendershot, Linda M

    2014-10-03

    ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

    Science.gov (United States)

    Chen, Qi; Luan, Zheng-Jiao; Yu, Hui-Lei; Cheng, Xiaolin; Xu, Jian-He

    2015-11-01

    A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Mycobacterium tuberculosis RecG binds and unwinds model DNA substrates with a preference for Holliday junctions.

    Science.gov (United States)

    Zegeye, Ephrem Debebe; Balasingham, Seetha V; Laerdahl, Jon K; Homberset, Håvard; Tønjum, Tone

    2012-08-01

    The RecG enzyme, a superfamily 2 helicase, is present in nearly all bacteria. Here we report for the first time that the recG gene is also present in the genomes of most vascular plants as well as in green algae, but is not found in other eukaryotes or archaea. The precise function of RecG is poorly understood, although ample evidence shows that it plays critical roles in DNA repair, recombination and replication. We further demonstrate that Mycobacterium tuberculosis RecG (RecG(Mtb)) DNA binding activity had a broad substrate specificity, whereas it only unwound branched-DNA substrates such as Holliday junctions (HJs), replication forks, D-loops and R-loops, with a strong preference for the HJ as a helicase substrate. In addition, RecG(Mtb) preferentially bound relatively long (≥40 nt) ssDNA, exhibiting a higher affinity for the homopolymeric nucleotides poly(dT), poly(dG) and poly(dC) than for poly(dA). RecG(Mtb) helicase activity was supported by hydrolysis of ATP or dATP in the presence of Mg(2+), Mn(2+), Cu(2+) or Fe(2+). Like its Escherichia coli orthologue, RecG(Mtb) is also a strictly DNA-dependent ATPase.

  13. Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

    Science.gov (United States)

    Thrower, J S; Blalock, R; Klinman, J P

    2001-08-14

    1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

  14. Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

    Science.gov (United States)

    Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

    2007-02-01

    The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

  15. Enhanced printability of thermoplastic polyurethane substrates by silica particles surface interactions

    Energy Technology Data Exchange (ETDEWEB)

    Cruz, S., E-mail: s.cruz@dep.uminho.pt [IPC/I3N – Institute of Polymers and Composites/Inst. of Nanostructures, Nanomodelling and Nanofabrication, Department Polymer Engineering, University of Minho, 4804-533 Guimarães (Portugal); Rocha, L.A. [CMEMS, University of Minho, 4804-533 Guimarães (Portugal); Viana, J.C. [IPC/I3N – Institute of Polymers and Composites/Inst. of Nanostructures, Nanomodelling and Nanofabrication, Department Polymer Engineering, University of Minho, 4804-533 Guimarães (Portugal)

    2016-01-01

    Graphical abstract: - Highlights: • A new method development for surface treatment of thermoplastic polyurethane (TPU) substrates. • The proposed method increases TPU surface energy (by 45%) and consequently the TPU wettability. • Great increase of the TPU surface roughness (by 621%). • Inkjet printed conductive ink was applied to the surface treated TPU substrate and significant improvements on the printability were obtained. - Abstract: A new method developed for the surface treatment of thermoplastic polymer substrates that increases their surface energies is introduced in this paper. The method is environmental friendly and low cost. In the proposed surface treatment method, nanoparticles are spread over the thermoplastic polyurethane (TPU) flexible substrate surface and then thermally fixed. This latter step allows the nanoparticles sinking-in on the polymer surface, resulting in a higher polymer–particle interaction at their interfacial region. The addition of nanoparticles onto the polymer surface increases surface roughness. The extent of the nanoparticles dispersion and sink-in in the substrate was evaluated through microscopy analysis (SEM). The roughness of the surface treated polymeric substrate was evaluated by AFM analysis. Substrate critical surface tension (ST) was measured by contact angle. In general, a homogeneous roughness form is achieved to a certain level. Great increase of the TPU surface roughness (by 621%) was induced by the propose method. The proposed surface treatment method increased significantly the substrate ST (by 45%) and consequently the TPU wettability. This novel surface treatment of thermoplastic polymers was applied to the inkjet printing of TPU substrates with conductive inks, and significant improvements on the printability were obtained.

  16. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

    Science.gov (United States)

    Broussard, Tyler C.; Pakhomova, Svetlana; Neau, David B.; Bonnot, Ross; Waldrop, Grover L.

    2015-01-01

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin. PMID:26020841

  17. Simulation of the fluid structure interaction for an aerostatic bearing and a flexible substrate

    NARCIS (Netherlands)

    Olieslagers, R.; Wild, M. de; Melick, S. van; Knaapen, R.

    2014-01-01

    The fluid structure interaction for an aerostatic bearing and a substrate is solved numerically by a semi-analytical model, programmed in the software package MATLAB. This semi-analytical model uses a fluidic network of resistances and capacities to solve the pressure field in the bearing channel.

  18. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

    Directory of Open Access Journals (Sweden)

    Mary C. Hames

    2013-11-01

    Full Text Available Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

  19. In vitro and in silico investigations of the binding interactions between chlorophenols and trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yan-Qing, E-mail: wyqing76@126.com [Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng City 224002, Jiangsu Province (China); Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China); Tan, Chun-Yun [Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China); Zhuang, Shu-Lin [Institute of Environmental Science, College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058 (China); Zhai, Peng-Zhan; Cui, Yun; Zhou, Qiu-Hua; Zhang, Hong-Mei [Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China); Fei, Zhenghao [Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng City 224002, Jiangsu Province (China); Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China)

    2014-08-15

    Graphical abstract: - Highlights: • Binding interactions of five chlorophenols with trypsin were investigated. • The number of chlorine atoms of chlorophenols partly affected the binding ability of them to trypsin. • Noncovalent interactions stabilized the trypsin–chlorophenols complexes. • There was the one main binding site of trypsin for chlorophenols. - Abstract: Being the first-degree toxic pollutants, chlorophenols (CP) have potential carcinogenic and mutagenic activity and toxicity. Since there still lacks studies on molecular interactions of chlorophenols with trypsin, one major binding target of many exogenous environmental pollutants, the binding interactions between five chlorophenols, 2-CP, 2,6-DCP, 2,4,6-TCP, 2,4,6-TCP, 2,3,4,6-TCP and PCP and trypsin were characterized by the combination of multispectroscopic techniques and molecular modeling. The chlorophenols bind at the one main site of trypsin and the binding induces the changes of microenvironment and global conformations of trypsin. Different number of chloride atoms significantly affects the binding and the binding constants K{sub A} ranks as K{sub A} (2-CP) < K{sub A} (2,6-DCP) ≈ K{sub A} (2,4,6-TCP) < K{sub A} (2,3,4,6-TCP) < K{sub A} (PCP). These chlorophenols interacts with trypsin mainly through hydrophobic interactions and via hydrogen bonding interactions and aromatic–aromatic π–π stacking interaction. Our results offer insights into the binding mechanism of chlorophenols with trypsin and provide important information for possible toxicity risk of chlorophenols to human health.

  20. Binding interaction between a queen pheromone component HOB and pheromone binding protein ASP1 of Apis cerana.

    Science.gov (United States)

    Weng, Chen; Fu, Yuxia; Jiang, Hongtao; Zhuang, Shulin; Li, Hongliang

    2015-01-01

    The honeybee's social behavior is closely related to the critical response to pheromone, while pheromone binding proteins (PBPs) play an important role in binding and transferring those pheromones. Here we report one known PBP, antennal special protein 1(ASP1), which has high affinity with a queen mandibular pheromone component, methyl-p-hydroxybenzoate (HOB). In this study, multiple fluorescent spectra, UV absorption spectra, circular dichroism (CD) spectra and molecular docking analysis were combined to clarify the binding process. Basically, fluorescence intensity of ASP1 could be considerably quenched by HOB with an appropriate interaction distance (3.1 nm), indicating that a complex, which is more stable in lower temperature, was formed. The fact ΔH < 0, ΔS < 0, by thermodynamic analysis, indicated the van der Waals and hydrogen bond as main driving force. Moreover, synchronous fluorescence spectra and CD spectra analysis showed the change of partial hydrophilicity of ASP1 and the increase of α-helix after HOB addition. In conclusion, ASP1 can strongly and spontaneously interact with HOB. But the binding ability decreases with the rise of temperature, which may be necessary for sufficient social stability of hives. This study provides elucidation of the detailed binding mechanism and potential physicochemical basis of thermal stability to the social behavior of honeybee. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2018-02-01

    The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

  2. Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

    Directory of Open Access Journals (Sweden)

    Magdalena Opalińska

    2017-11-01

    Full Text Available Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4 and Pam18-2 and known (Tim17-2 substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

  3. Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly.

    Science.gov (United States)

    Davies, Brian A; Azmi, Ishara F; Payne, Johanna; Shestakova, Anna; Horazdovsky, Bruce F; Babst, Markus; Katzmann, David J

    2010-10-01

    ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

  4. Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

    DEFF Research Database (Denmark)

    Wierzbicki, Rafal; Købler, Carsten; Jensen, Mikkel Ravn Boye

    2013-01-01

    Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold...... substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition...

  5. An autoradiographic map of (3H)diprenorphine binding in rat brain: effects of social interaction

    International Nuclear Information System (INIS)

    Panksepp, J.; Bishop, P.

    1981-01-01

    (3H)Diprenorphine binding was analyzed autoradiographically in the brains of 33 day old rat pups. A photographic atlas of diprenorphine binding in the coronal plane is provided to highlight the dispersion of opioid receptor systems through the brain. To determine whether brain opioid release may be induced by social interactions, half the animals were sacrificed following a 30 min period of social interaction while the other half were sacrificed following 30 min of social isolation. Opioid binding was higher in isolate-tested animals than socially-tested ones, suggesting that social interaction may promote endogenous brain opioid release

  6. Thermodynamics of binding interactions between extracellular polymeric substances and heavy metals by isothermal titration microcalorimetry.

    Science.gov (United States)

    Yan, Peng; Xia, Jia-Shuai; Chen, You-Peng; Liu, Zhi-Ping; Guo, Jin-Song; Shen, Yu; Zhang, Cheng-Cheng; Wang, Jing

    2017-05-01

    Extracellular polymeric substances (EPS) play a crucial role in heavy metal bio-adsorption using activated sludge, but the interaction mechanism between heavy metals and EPS remains unclear. Isothermal titration calorimetry was employed to illuminate the mechanism in this study. The results indicate that binding between heavy metals and EPS is spontaneous and driven mainly by enthalpy change. Extracellular proteins in EPS are major participants in the binding process. Environmental conditions have significant impact on the adsorption performance. Divalent and trivalent cations severely impeded the binding of heavy metal ions to EPS. Electrostatic interaction mainly attributed to competition between divalent cations and heavy metal ions; trivalent cations directly competed with heavy metal ions for EPS binding sites. Trivalent cations were more competitive than divalent cations for heavy metal ion binding because they formed complexing bonds. This study facilitates a better understanding about the interaction between heavy metals and EPS in wastewater treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The use of LeuT as a model in elucidating binding sites for substrates and inhibitors in neurotransmitter transporters

    DEFF Research Database (Denmark)

    Løland, Claus Juul

    2015-01-01

    Background: The mammalian neurotransmitter transporters are complex proteins playing a central role in synaptic transmission between neurons by rapid reuptake of neurotransmitters. The proteins which transport dopamine, noradrenaline and serotonin belong to the Neurotransmitter:Sodium Symporters...... (NSS). Due to their important role, dysfunctions are associated with several psychiatric and neurological diseases and they also serve as targets for a wide range of therapeutic and illicit drugs. Despite the central physiological and pharmacological importance, direct evidence on structure......–function relationships on mammalian NSS proteins has so far been unsuccessful. The crystal structure of the bacterial NSS protein, LeuT, has been a turning point in structural investigations. Scope of review: To provide an update on what is known about the binding sites for substrates and inhibitors in the Leu...

  8. Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis.

    Science.gov (United States)

    Kulminskaya, Anna A; Arand, Michael; Eneyskaya, Elena V; Ivanen, Dina R; Shabalin, Konstantin A; Shishlyannikov, Sergei M; Saveliev, Andrew N; Korneeva, Olga S; Neustroev, Kirill N

    2003-08-21

    1H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity. Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme hydrolyzed both beta2,1- as well as beta2,6-fructosyl linkages in fructooligosaccharides. From the k(cat)/K(m) ratios obtained with inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase.

  9. Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

    Science.gov (United States)

    Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

    2003-08-01

    Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

  10. Thermal interaction between WC-Co coating and steel substrate in process of HVOF spraying

    International Nuclear Information System (INIS)

    Guilemany, J.M.; Sobolev, V.V.; Nutting, J.; Dong, Z.; Calero, J.A.

    1994-01-01

    The WC-Co powders can be used to produce good adhesive and wear resistant HVOF thermal spray coatings on steel and light alloys substrates. In order to understand the properties of this kind of coating, the phases which are present in the coatings and structure changes during post heat treatments have been investigated. Although the coating properties depend very much on the structure developed in the substrate-coating interfacial region it has not been yet investigated in detail. The present study is devoted to the experimental and theoretical analysis of this interfacial region. The structure characterization has been performed mainly through the use of transmission electron microscopy. To provide a theoretical investigation a realistic prediction model of the process has been developed and on its base the mathematical simulation of the substrate-coating thermal interaction has been undertaken

  11. Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation

    NARCIS (Netherlands)

    Afdhal, N. H.; Ostrow, J. D.; Koehler, R.; Niu, N.; Groen, A. K.; Veis, A.; Nunes, D. P.; Offner, G. D.

    1995-01-01

    Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4

  12. Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

    Science.gov (United States)

    Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

    2017-11-01

    P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

  13. Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

    Science.gov (United States)

    Krishnamoorthy, Ezhilarasi; Hassan, Sameer; Hanna, Luke Elizabeth; Padmalayam, Indira; Rajaram, Rama; Viswanathan, Vijay

    2017-05-07

    Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features.

    Directory of Open Access Journals (Sweden)

    Renata De Paris

    Full Text Available Protein receptor conformations, obtained from molecular dynamics (MD simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids and four agglomerative hierarchical methods (Complete linkage, Ward's, Unweighted Pair Group Method and Weighted Pair Group Method to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to

  15. Substrate specificity, metal binding properties, and spectroscopic characterization of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae.

    Science.gov (United States)

    Bienvenue, David L; Gilner, Danuta M; Davis, Ryan S; Bennett, Brian; Holz, Richard C

    2003-09-16

    The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to approximately 20% of the Zn(II)-loaded enzyme. The order of the observed k(cat) values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) approximately equal Cu(II) approximately equal Mg(II). DapE was shown to only hydrolyze L,L-N-succinyl-diaminopimelic acid (L,L-SDAP) and was inactive toward D,L-, L,D-, and D,D-SDAP. DapE was also inactive toward several acetylated amino acids as well as D,L-succinyl aminopimelate, which differs from the natural substrate, L,L-SDAP, by the absence of the amine group on the amino acid side chain. These data imply that the carboxylate of the succinyl moiety and the amine form important interactions with the active site of DapE. The affinity of DapE for one versus two Zn(II) ions differs by nearly 2.2 x 10(3) times (K(d1) = 0.14 microM vs K(d2) = 300 microM). In addition, an Arrhenius plot was constructed from k(cat) values measured between 16 and 35 degrees C and was linear over this temperature range. The activation energy for [ZnZn(DapE)] was found to be 31 kJ/mol with the remaining thermodynamic parameters calculated at 25 degrees C being DeltaG(++) = 64 kJ/mol, DeltaH(++) = 28.5 kJ/mol, and DeltaS(++) = -119 J mol(-1) K(-1). Electronic absorption and EPR spectra of [Co_(DapE)] and [CoCo(DapE)] indicate that the first Co(II) binding site is five-coordinate, while the second site is octahedral. In addition, any spin-spin interaction between the two Co(II) ions in [CoCo(DapE)] is very weak. The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and

  16. Synchrotron radiation in the Far-Infrared: Adsorbate-substrate vibrations and resonant interactions

    International Nuclear Information System (INIS)

    Hoffmann, F.M.; Williams, G.P.; Hirschmugl, C.J.; Chabal, Y.J.

    1991-01-01

    Synchrotron radiation in the Far Infrared offers the potential for a broadband source of high brightness and intensity. Recent development of a Far-Infrared Beamline at the NSLS in Brookhaven provides an unique high intensity source in the FIR spectral range (800-10 cm -1 ). This talk reviews its application to surface vibrational spectroscopy of low frequency adsorbate-substrate vibrations and resonant interactions on metal surfaces

  17. Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Keishi Yamasaki

    Full Text Available A wide variety of drugs bind to human serum albumin (HSA at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand and ibuprofen (site II ligand at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a 'competitive-like' allosteric interaction in the case of the defatted HSA conformer to a 'nearly independent' binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a 'competitive-like' allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to 'nearly independent' binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

  18. Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2.

    Directory of Open Access Journals (Sweden)

    Roche C de Guzman

    Full Text Available Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2 has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD of 1.8 × 10(-4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7 M, within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5 M. BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks, suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5, below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the

  19. Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump

    International Nuclear Information System (INIS)

    Vorherr, T.; James, P.; Krebs, J.; Carafoli, E.; McCormick, D.J.; Penniston, J.T.; Enyedi, A.

    1990-01-01

    Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca 2+ pump were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca 2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with calmodulin binding domain, supporting the suggestion that the latter, or a portion of it, may act as a natural inhibitor of the pump

  20. Binding interactions between suberin monomer components and pesticides

    Energy Technology Data Exchange (ETDEWEB)

    Olivella, M.À., E-mail: angels.olivella@udg.edu [Department of Chemical Engineering, Escola Politècnica Superior, Universitat de Girona, Maria Aurèlia Capmany, 61, 17071 Girona (Spain); Bazzicalupi, C.; Bianchi, A. [Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia, 3, 50019 Sesto Fiorentino (Italy); Río, J.C. del [Instituto de Recursos Naturales y Agrobiología de Sevilla, Consejo Superior de Investigaciones Científicas, P.O. Box 1052, 41080 Seville (Spain); Fiol, N.; Villaescusa, I. [Department of Chemical Engineering, Escola Politècnica Superior, Universitat de Girona, Maria Aurèlia Capmany, 61, 17071 Girona (Spain)

    2015-09-15

    Understanding the role of biomacromolecules and their interactions with pollutants is a key for elucidating the sorption mechanisms and making an accurate assessment of the environmental fate of pollutants. The knowledge of the sorption properties of the different constituents of these biomacromolecules may furnish a significant contribution to this purpose. Suberin is a very abundant biopolymer in higher plants. In this study, suberin monomers isolated from cork were analyzed by thermally-assisted methylation with tetramethylammonium hydroxide (TMAH) in a pyrolysis unit coupled to gas chromatography–mass spectrometry (GC/MS). The isolated monomer mixture was used to study the sorption of three pesticides (isoproturon, methomyl and oxamyl). The modes of pesticide–sorbent interactions were analyzed by means of two modeling calculations, the first one representing only the mixture of suberin monomers used in the sorption study, and the second one including glycerol to the mixture of suberin monomers, as a building block of the suberin molecule. The results indicated that the highest sorption capacity exhibited by the sorbent was for isoproturon (33%) being methomyl and oxamyl sorbed by the main suberin components to a lesser extent (3% and < 1%, respectively). In addition to van der Waals interactions with the apolar region of sorbent and isoproturon, modeling calculations evidenced the formation of a hydrogen bond between the isoproturon NH group and a carboxylic oxygen atom of a suberin monomer. In the case of methomyl and oxamyl only weak van der Waals interactions stabilize the pesticide–sorbent adducts. The presence of glycerol in the model provoked significant changes in the interactions with isoproturon and methomyl. - Highlights: • Suberin has low affinity to retain pesticides of aliphatic character. • Suberin has a moderate affinity to adsorb isoproturon. • Modeling calculations show that apolar portion of suberin interacts with isoproturon.

  1. Binding interactions between suberin monomer components and pesticides

    International Nuclear Information System (INIS)

    Olivella, M.À.; Bazzicalupi, C.; Bianchi, A.; Río, J.C. del; Fiol, N.; Villaescusa, I.

    2015-01-01

    Understanding the role of biomacromolecules and their interactions with pollutants is a key for elucidating the sorption mechanisms and making an accurate assessment of the environmental fate of pollutants. The knowledge of the sorption properties of the different constituents of these biomacromolecules may furnish a significant contribution to this purpose. Suberin is a very abundant biopolymer in higher plants. In this study, suberin monomers isolated from cork were analyzed by thermally-assisted methylation with tetramethylammonium hydroxide (TMAH) in a pyrolysis unit coupled to gas chromatography–mass spectrometry (GC/MS). The isolated monomer mixture was used to study the sorption of three pesticides (isoproturon, methomyl and oxamyl). The modes of pesticide–sorbent interactions were analyzed by means of two modeling calculations, the first one representing only the mixture of suberin monomers used in the sorption study, and the second one including glycerol to the mixture of suberin monomers, as a building block of the suberin molecule. The results indicated that the highest sorption capacity exhibited by the sorbent was for isoproturon (33%) being methomyl and oxamyl sorbed by the main suberin components to a lesser extent (3% and < 1%, respectively). In addition to van der Waals interactions with the apolar region of sorbent and isoproturon, modeling calculations evidenced the formation of a hydrogen bond between the isoproturon NH group and a carboxylic oxygen atom of a suberin monomer. In the case of methomyl and oxamyl only weak van der Waals interactions stabilize the pesticide–sorbent adducts. The presence of glycerol in the model provoked significant changes in the interactions with isoproturon and methomyl. - Highlights: • Suberin has low affinity to retain pesticides of aliphatic character. • Suberin has a moderate affinity to adsorb isoproturon. • Modeling calculations show that apolar portion of suberin interacts with isoproturon.

  2. Combined fluorescence and electrochemical investigation on the binding interaction between organic acid and human serum albumin

    Institute of Scientific and Technical Information of China (English)

    CHEN Yan-Min; GUO Liang-Hong

    2009-01-01

    Human serum albumin (HSA) is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME (absorption, distribution, metabolism, and excretion) and bioavailability of the pollutants. We report investigation on the binding interaction between HSA and acetic acid (C2), octanoic acid (C8) and dodecanoic acid (C12) by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. Two fluorescent probes, dansylamide and dansyl-L-proline, were employed in the displacement measurement to study fatty acid interaction with the two drug-binding sites on HSA. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations made by the three approaches are very similar. HSA did not show any change in either fluorescence or electrochemistry after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential fashion, indicative of independent and non-cooperative binding. The calculated association constant and binding ratio is 3.1×106 L/mol and 1 with drug binding Site I, 1.1×107 L/mol and 1 with Site II, and 7.0×104 L/mol and 4 with the tryptophan site. The measurement with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.

  3. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-01-01

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

  4. Dissecting Hofmeister Effects: Direct Anion-Amide Interactions Are Weaker than Cation-Amide Binding.

    Science.gov (United States)

    Balos, Vasileios; Kim, Heejae; Bonn, Mischa; Hunger, Johannes

    2016-07-04

    Whereas there is increasing evidence for ion-induced protein destabilization through direct ion-protein interactions, the strength of the binding of anions to proteins relative to cation-protein binding has remained elusive. In this work, the rotational mobility of a model amide in aqueous solution was used as a reporter for the interactions of different anions with the amide group. Protein-stabilizing salts such as KCl and KNO3 do not affect the rotational mobility of the amide. Conversely, protein denaturants such as KSCN and KI markedly reduce the orientational freedom of the amide group. Thus these results provide evidence for a direct denaturation mechanism through ion-protein interactions. Comparing the present findings with results for cations shows that in contrast to common belief, anion-amide binding is weaker than cation-amide binding. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide.

    Science.gov (United States)

    Bathaie, S Z; Moosavi-Movahedi, A A; Saboury, A A

    1999-02-15

    The interaction of dodecyl trimethylammonium bromide (DTAB), a cationic surfactant, with calf thymus DNA has been studied by various methods, including potentiometric technique using DTAB-selective plastic membrane electrode at 27 and 37 degreesC, isothermal titration microcalorimetry and UV spectrophotometry at 27 degreesC using 0.05 M Tris buffer and 0.01 M NaCl at pH 7.4. The free energy is calculated from binding isotherms on the basis of Wyman binding potential theory and the enthalpy of binding according to van't Hoff relation. The enthalpy of unfolding has been determined by subtraction of the enthalpy of binding from the microcalorimetric enthalpy. The results show that, after the interaction of first DTAB molecule to DNA (base molarity) through the electrostatic interaction, the second DTAB molecule also binds to DNA through electrostatic interaction. At this stage, the predom-inant DNA conformational change occurs. Afterwards up to 20 DTAB molecules, below the critical micelle concentration of DTAB, bind through hydrophobic interactions.

  6. Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

    Science.gov (United States)

    Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

    2018-06-16

    Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

  7. Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β-Lactamases.

    Science.gov (United States)

    Hargis, Jacqueline C; Vankayala, Sai Lakshmana; White, Justin K; Woodcock, H Lee

    2014-02-11

    Bacterial resistance to standard (i.e., β-lactam-based) antibiotics has become a global pandemic. Simultaneously, research into the underlying causes of resistance has slowed substantially, although its importance is universally recognized. Key to unraveling critical details is characterization of the noncovalent interactions that govern binding and specificity (DD-peptidases, antibiotic targets, versus β-lactamases, the evolutionarily derived enzymes that play a major role in resistance) and ultimately resistance as a whole. Herein, we describe a detailed investigation that elicits new chemical insights into these underlying intermolecular interactions. Benzylpenicillin and a novel β-lactam peptidomimetic complexed to the Stremptomyces R61 peptidase are examined using an arsenal of computational techniques: MD simulations, QM/MM calculations, charge perturbation analysis, QM/MM orbital analysis, bioinformatics, flexible receptor/flexible ligand docking, and computational ADME predictions. Several key molecular level interactions are identified that not only shed light onto fundamental resistance mechanisms, but also offer explanations for observed specificity. Specifically, an extended π-π network is elucidated that suggests antibacterial resistance has evolved, in part, due to stabilizing aromatic interactions. Additionally, interactions between the protein and peptidomimetic substrate are identified and characterized. Of particular interest is a water-mediated salt bridge between Asp217 and the positively charged N-terminus of the peptidomimetic, revealing an interaction that may significantly contribute to β-lactam specificity. Finally, interaction information is used to suggest modifications to current β-lactam compounds that should both improve binding and specificity in DD-peptidases and their physiochemical properties.

  8. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    Science.gov (United States)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  9. The Complement Binding and Inhibitory Protein CbiA of Borrelia miyamotoi Degrades Extracellular Matrix Components by Interacting with Plasmin(ogen

    Directory of Open Access Journals (Sweden)

    Ngoc T. T. Nguyen

    2018-02-01

    Full Text Available The emerging relapsing fever spirochete Borrelia (B. miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD. More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen. Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.

  10. Hypernuclear interactions and the binding energies of and hypernuclei

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R.; Usmani, Q.N.

    1988-01-01

    By use of variational calculations a reasonable hadronic description is obtained of the s-shell hypernuclei, of /sub /ZBe, and of the well depth, with N forces which are consistent with p scattering and which are quite strongly spin-dependent, with reasonable TPE NN forces with strongly repulsive dispersive-type NN forces. For the latter we also consider a spin-dependent version which is somewhat favored by our analysis. /sub /ZBe is treated as a 2ed + system and is significantly overbound, approx. =1 MeV, if only ed ed and ed potentials are used. An ed ed potential obtained from the NN forces nicely accounts for this overbinding. The hypernuclei /sub /WHe and /sub / Be are treated as ed + 2 and 2ed + 2 systems. Use of the /sub / Be event gives approx. =1.5 MeV too little binding for /sub /WHe. The S0 potential obtained from /sub / Be is quite strongly attractive, comparable to the N and also to the NN potential without OPE. 18 refs.

  11. Binding energies of hypernuclei and Λ-nuclear interactions

    International Nuclear Information System (INIS)

    Bodmer, A.R.; Usmani, Q.N.

    1984-01-01

    Variational calculations indicate that a reasonable description of Λp scattering and of Λ separation energies can be obtained in terms of ΛN plus dispersive and TPE ΛNN forces. Results for the ΛΛ interaction and for 6 /sub Λ/He obtained from an analysis of 10 /sub ΛΛ/Be are discussed. Coulomb and ΛN charge symmetry breaking effects in the A = 4 hypernuclei are discussed

  12. Root development during soil genesis: effects of root-root interactions, mycorrhizae, and substrate

    Science.gov (United States)

    Salinas, A.; Zaharescu, D. G.

    2015-12-01

    A major driver of soil formation is the colonization and transformation of rock by plants and associated microbiota. In turn, substrate chemical composition can also influence the capacity for plant colonization and development. In order to better define these relationships, a mesocosm study was set up to analyze the effect mycorrhizal fungi, plant density and rock have on root development, and to determine the effect of root morphology on weathering and soil formation. We hypothesized that plant-plant and plant-fungi interactions have a stronger influence on root architecture and rock weathering than the substrate composition alone. Buffalo grass (Bouteloua dactyloides) was grown in a controlled environment in columns filled with either granular granite, schist, rhyolite or basalt. Each substrate was given two different treatments, including grass-microbes and grass-microbes-mycorrhizae and incubated for 120, 240, and 480 days. Columns were then extracted and analyzed for root morphology, fine fraction, and pore water major element content. Preliminary results showed that plants produced more biomass in rhyolite, followed by schist, basalt, and granite, indicating that substrate composition is an important driver of root development. In support of our hypothesis, mycorrhizae was a strong driver of root development by stimulating length growth, biomass production, and branching. However, average root length and branching also appeared to decrease in response to high plant density, though this trend was only present among roots with mycorrhizal fungi. Interestingly, fine fraction production was negatively correlated with average root thickness and volume. There is also slight evidence indicating that fine fraction production is more related to substrate composition than root morphology, though this data needs to be further analyzed. Our hope is that the results of this study can one day be applied to agricultural research in order to promote the production of crops

  13. Sudden cardiac death: the pro-arrhythmic interaction of an acute loading with an underlying substrate.

    Science.gov (United States)

    Sutherland, George R

    2017-10-21

    Sudden cardiac death (SCD) is a complex phenomenon, occurring either in apparently normal individuals or in those where there is a recognized underlying cardiac abnormality. In both groups, the lethal arrhythmia has frequently been related to the physiologic trigger of either exercise or stress. Prior research into SCD has focused mainly on a combination of identifying either vulnerable myocardial substrates; pharmacological approaches to altering electrical activation/repolarisation in substrates; or the suppression of induced lethal arrhythmias with implantable defibrillators. However, it has been suggested that in a significant number of cases, the interaction of a transient induced trigger with a pre-existing electrical or mechanical substrate is the basis for the induction of the sustained lethal arrhythmia. In this manuscript we will discuss the precise mechanisms whereby one of such potential physiologic trigger: an acute change in systolic blood pressure, can induce a sequence of alterations in global and local cardiac mechanics which in turn result in regional left ventricular post-systolic deformation which, mediated (through stretch-induced changes in local mechano-electrical coupling) provokes local electrical after-depolarisations which can spill over into complex runs of premature ventricular beats. These local acute pressure/stretch induced runs of ventricular ectopy originate in either basal or apical normal myocardium and, in combination with a co-existing distal pro-arrhymic substrate, can interact to induce a lethal arrhythmia. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For Permissions, please email: journals.permissions@oup.com.

  14. How Are Substrate Binding and Catalysis Affected by Mutating Glu127 and Arg161 in Prolyl-4-hydroxylase? A QM/MM and MD Study

    Science.gov (United States)

    Timmins, Amy; de Visser, Sam P.

    2017-01-01

    Prolyl-4-hydroxylase is a vital enzyme for human physiology involved in the biosynthesis of 4-hydroxyproline, an essential component for collagen formation. The enzyme performs a unique stereo- and regioselective hydroxylation at the C4 position of proline despite the fact that the C5 hydrogen atoms should be thermodynamically easier to abstract. To gain insight into the mechanism and find the origin of this regioselectivity, we have done a quantum mechanics/molecular mechanics (QM/MM) study on wildtype and mutant structures. In a previous study (Timmins et al., 2017) we identified several active site residues critical for substrate binding and positioning. In particular, the Glu127 and Arg161 were shown to form multiple hydrogen bonding and ion-dipole interactions with substrate and could thereby affect the regio- and stereoselectivity of the reaction. In this work, we decided to test that hypothesis and report a QM/MM and molecular dynamics (MD) study on prolyl-4-hydroxylase and several active site mutants where Glu127 or Arg161 are mutated for Asp, Gln, or Lys. Thus, the R161D and R161Q mutants give very high barriers for hydrogen atom abstraction from any proline C–H bond and therefore will be inactive. The R161K mutant, by contrast, sees the regio- and stereoselectivity of the reaction change but still is expected to hydroxylate proline at room temperature. By contrast, the Glu127 mutants E127D and E127Q show possible changes in regioselectivity with the former being more probable to react compared to the latter. PMID:29170737

  15. The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

    Directory of Open Access Journals (Sweden)

    Ruth M Kennan

    Full Text Available Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by

  16. How Are Substrate Binding and Catalysis Affected by Mutating Glu127 and Arg161 in Prolyl-4-hydroxylase? A QM/MM and MD Study

    Directory of Open Access Journals (Sweden)

    Amy Timmins

    2017-11-01

    Full Text Available Prolyl-4-hydroxylase is a vital enzyme for human physiology involved in the biosynthesis of 4-hydroxyproline, an essential component for collagen formation. The enzyme performs a unique stereo- and regioselective hydroxylation at the C4 position of proline despite the fact that the C5 hydrogen atoms should be thermodynamically easier to abstract. To gain insight into the mechanism and find the origin of this regioselectivity, we have done a quantum mechanics/molecular mechanics (QM/MM study on wildtype and mutant structures. In a previous study (Timmins et al., 2017 we identified several active site residues critical for substrate binding and positioning. In particular, the Glu127 and Arg161 were shown to form multiple hydrogen bonding and ion-dipole interactions with substrate and could thereby affect the regio- and stereoselectivity of the reaction. In this work, we decided to test that hypothesis and report a QM/MM and molecular dynamics (MD study on prolyl-4-hydroxylase and several active site mutants where Glu127 or Arg161 are mutated for Asp, Gln, or Lys. Thus, the R161D and R161Q mutants give very high barriers for hydrogen atom abstraction from any proline C–H bond and therefore will be inactive. The R161K mutant, by contrast, sees the regio- and stereoselectivity of the reaction change but still is expected to hydroxylate proline at room temperature. By contrast, the Glu127 mutants E127D and E127Q show possible changes in regioselectivity with the former being more probable to react compared to the latter.

  17. Analysis of the spectroscopic characteristics on the binding interaction between tosufloxacin and bovine lactoferrin

    International Nuclear Information System (INIS)

    Guo Ming; Zhang Luying; Lue Weijun; Cao Huaru

    2011-01-01

    The interaction between tosufloxacin (TELX) and bovine lactoferrin (BLF) in aqueous solution was analyzed by fluorescence spectroscopy and absorbance spectra. The binding parameters and energy-transfer efficiency parameters were determined and the mechanism of interaction was discussed. The effect of tosufloxacin acting on the BLF's conformation was detected and the unfolding procedure of BLF induced by tosufloxacin was explored by 'fluorescence phase diagram'. Following experimental data of fluorescence polarization values P and r, the saturation characteristic of such kind of binding reaction was observed for the first time. The interaction between tosufloxacin and BLF influenced by Ni 2+ and Co 2+ were also preliminarily explored in this work. - Research Highlights: →In this paper, a new saturation spectroscopic characteristic of non-covalent binding reaction is proposed. The saturated character of interaction of tosufloxacin binding with bovine lactoferrin is firstly observed by fluorescence polarization spectroscopy. →The unfolding procedure of bovine lactoferrin induced by drug ligand is analyzed by 'fluorescence phase diagram', and it is quantitatively characterized. →The binding parameters and energy-transfer efficiency parameters of bovine lactoferrin-tosufloxacin/tosufloxacin-Co 2+ (Ni 2+ ) system are determined and the mechanism of interaction is discussed.

  18. Spectrophotometric analysis of flavonoid-DNA binding interactions at physiological conditions

    Science.gov (United States)

    Janjua, Naveed Kausar; Siddiqa, Asima; Yaqub, Azra; Sabahat, Sana; Qureshi, Rumana; Haque, Sayed ul

    2009-12-01

    Mode of interactions of three flavonoids [morin (M), quercetin (Q), and rutin (R)] with chicken blood ds.DNA (ck.DNA) has been investigated spectrophotometrically at different temperatures including body temperature (310 K) and at two physiological pH values, i.e. 7.4 (human blood pH) and 4.7 (stomach pH). The binding constants, Kf, evaluated using Benesi-Hildebrand equation showed that the flavonoids bind effectively through intercalation at both pH values and body temperature. Quercetin, somehow, showed greater binding capabilities with DNA. The free energies of flavonoid-DNA complexes indicated the spontaneity of their binding. The order of binding constants of three flavonoids at both pH values were found to be Kf(Q) > Kf(R) > Kf(M) and at 310 K.

  19. Configuration interaction calculations of positron binding to Be({sup 3}P )

    Energy Technology Data Exchange (ETDEWEB)

    Bromley, M.W.J. [Department of Physics, San Diego State University, San Diego, CA 92182 (United States)]. E-mail: mbromley@physics.sdsu.edu; Mitroy, J. [Faculty of Technology, Charles Darwin University, Darwin, NT 0909 (Australia)]. E-mail: jxm107@rsphysse.anu.edu.au

    2006-06-15

    The configuration interaction method is applied to investigate the possibility of positron binding to the metastable beryllium (1s{sup 2}2s2p {sup 3}P ) state. The largest calculation obtained an estimated energy that was unstable by 0.00014 Hartree with respect to the Ps + Be{sup +}(2s) lowest dissociation channel. It is likely that positron binding to parent states with non-zero angular momentum is inhibited by centrifugal barriers.

  20. Theoretical study of the interparticle interaction of nanoparticles randomly dispersed on a substrate

    International Nuclear Information System (INIS)

    Horikoshi, S.; Kato, T.

    2015-01-01

    Metal nanoparticles exhibit the phenomenon of localized surface plasmon resonance (LSPR) due to the collective oscillation of their conduction electrons, which is induced by external electromagnetic radiation. The finite-differential time-domain (FDTD) method is widely used as an electromagnetic field analysis tool for nanoparticles. Although the influence of interparticle interactions is taken into consideration in the FDTD calculation for the plural particles configuration, the FDTD calculation of a random configuration is very difficult, particularly in the case of non-spherical particles. In this study, a theoretical calculation method incorporating interparticle interactions on a substrate with various particle shapes and sizes on a subwavelength scale is developed. The interparticle interaction is incorporated following FDTD calculation with an isolated single particle. This is explained systematically using a signal flow graph. Moreover, the mirror image effect of the substrate and the retardation effect are also taken into account in this method. The validity of this method is verified by calculations for simple arrangements of nanoparticles. In addition, it is confirmed that the method can improve the accuracy of predicted experimental results for Au nanoparticles prepared by the sputtering method, in terms of the plasmon peak wavelength. This method may enable the design of LSPR devices by controlling nanoparticle characteristics, such as the size, shape, and distribution density

  1. Ultrafast dynamics of ligand and substrate interaction in endothelial nitric oxide synthase under Soret excitation.

    Science.gov (United States)

    Hung, Chih-Chang; Yabushita, Atsushi; Kobayashi, Takayoshi; Chen, Pei-Feng; Liang, Keng S

    2016-01-01

    Ultrafast transient absorption spectroscopy of endothelial NOS oxygenase domain (eNOS-oxy) was performed to study dynamics of ligand or substrate interaction under Soret band excitation. Photo-excitation dissociates imidazole ligand in 4ps. The eNOS-oxy without additive is partially bound with water molecule, thus its photoexcited dynamics also shows ligand dissociation in <800fs. Then it followed by vibrational cooling coupled with charge transfer in 4.8ps, and recombination of ligand to distal side of heme in 12ps. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Binding energies of hypernuclei and Λ-nuclear interactions

    International Nuclear Information System (INIS)

    Bodmer, A.R.; Usmani, Q.N.

    1985-01-01

    Variational Monte Carlo calculations have been made for the s-shell hypernuclei and also of 9 Be hypernuclei with a 2α + Λ model. The well depth is calculated variationally with the Fermi hypernetted chain method. A satisfactory description of all the relevant experimental Λ separation energies and also of the Λp scattering can be obtained with reasonable TPE ΛN and ΛNN forces and strongly repulsive dispersive ΛNN forces which are preferred to be spin dependent. We discuss variational calculations for 6 He and 10 Be hypernuclei with α + 2Λ and 2α + 2Λ models, and the results obtained for the ΛΛ interaction and for 6 He hypernuclei from analysis of 10 Be hypernuclei Coulomb effects and charge symmetry breaking in the A = 4 hypernuclei are discussed. 24 refs., 5 figs

  3. Binding energies of hypernuclei and. lambda. -nuclear interactions

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R.; Usmani, Q.N.

    1985-01-01

    Variational Monte Carlo calculations have been made for the s-shell hypernuclei and also of /sup 9/Be hypernuclei with a 2..cap alpha.. + ..lambda.. model. The well depth is calculated variationally with the Fermi hypernetted chain method. A satisfactory description of all the relevant experimental ..lambda.. separation energies and also of the ..lambda..p scattering can be obtained with reasonable TPE ..lambda..N and ..lambda..NN forces and strongly repulsive dispersive ..lambda..NN forces which are preferred to be spin dependent. We discuss variational calculations for /sup 6/He and /sup 10/Be hypernuclei with ..cap alpha.. + 2..lambda.. and 2..cap alpha.. + 2..lambda.. models, and the results obtained for the ..lambda lambda.. interaction and for /sup 6/He hypernuclei from analysis of /sup 10/Be hypernuclei Coulomb effects and charge symmetry breaking in the A = 4 hypernuclei are discussed. 24 refs., 5 figs.

  4. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    Energy Technology Data Exchange (ETDEWEB)

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

  5. Interactions of poly(amidoamine) dendrimers with human serum albumin: binding constants and mechanisms.

    Science.gov (United States)

    Giri, Jyotsnendu; Diallo, Mamadou S; Simpson, André J; Liu, Yi; Goddard, William A; Kumar, Rajeev; Woods, Gwen C

    2011-05-24

    The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K(b)) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To gain insight into the mechanisms of HSA binding to PAMAM dendrimers, we combined (1)H NMR, saturation transfer difference (STD) NMR, and NMR diffusion ordered spectroscopy (DOSY) of dendrimer-HSA complexes with atomistic molecular dynamics (MD) simulations of dendrimer conformation in aqueous solutions. The binding measurements show that the HSA binding constants (K(b)) of PAMAM dendrimers depend on dendrimer size and terminal group chemistry. The NMR (1)H and DOSY experiments indicate that the interactions between HSA and PAMAM dendrimers are relatively weak. The (1)H NMR STD experiments and MD simulations suggest that the inner shell protons of the dendrimers groups interact more strongly with HSA proteins. These interactions, which are consistently observed for different dendrimer generations (G0-NH(2)vs G4-NH(2)) and terminal groups (G4-NH(2)vs G4-OH with amidoethanol groups), suggest that PAMAM dendrimers adopt backfolded configurations as they form weak complexes with HSA proteins in aqueous solutions at physiological pH (7.4).

  6. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Carson M Andorf

    Full Text Available Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners.Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques.Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions.We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

  7. Evaluation of Selected CYP51A1 Polymorphisms in View of Interactions with Substrate and Redox Partner

    Directory of Open Access Journals (Sweden)

    Tadeja Režen

    2017-06-01

    Full Text Available Cholesterol is essential for development, growth, and maintenance of organisms. Mutations in cholesterol biosynthetic genes are embryonic lethal and few polymorphisms have been so far associated with pathologies in humans. Previous analyses show that lanosterol 14α-demethylase (CYP51A1 from the late part of cholesterol biosynthesis has only a few missense mutations with low minor allele frequencies and low association with pathologies in humans. The aim of this study is to evaluate the role of amino acid changes in the natural missense mutations of the hCYP51A1 protein. We searched SNP databases for existing polymorphisms of CYP51A1 and evaluated their effect on protein function. We found rare variants causing detrimental missense mutations of CYP51A1. Some missense variants were also associated with a phenotype in humans. Two missense variants have been prepared for testing enzymatic activity in vitro but failed to produce a P450 spectrum. We performed molecular modeling of three selected missense variants to evaluate the effect of the amino acid substitution on potential interaction with its substrate and the obligatory redox partner POR. We show that two of the variants, R277L and especially D152G, have possibly lower binding potential toward obligatory redox partner POR. D152G and R431H have also potentially lower affinity toward the substrate lanosterol. We evaluated the potential effect of damaging variants also using data from other in vitro CYP51A1 mutants. In conclusion, we propose to include damaging CYP51A1 variants into personalized diagnostics to improve genetic counseling for certain rare disease phenotypes.

  8. Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

    Science.gov (United States)

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

    2016-09-13

    Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

  9. The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

    Science.gov (United States)

    Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

    2017-08-01

    The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

    Science.gov (United States)

    Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

    2018-04-01

    Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

  11. Endocrine disruption: In silico interactions between phthalate plasticizers and corticosteroid binding globulin.

    Science.gov (United States)

    Sheikh, Ishfaq A; Beg, Mohd A

    2017-12-01

    Endocrine disruption is a phenomenon when a man-made or natural compound interferes with normal hormone function in human or animal body systems. Endocrine-disrupting compounds (EDCs) have assumed considerable importance as a result of industrial activity, mass production of synthetic chemicals and environmental pollution. Phthalate plasticizers are a group of chemicals used widely and diversely in industry especially in the plastic industry, and many of the phthalate compounds have endocrine-disrupting properties. Increasing evidence indicates that steroid nuclear receptors and steroid binding proteins are the main targets of endocrine disruption. Corticosteroid-binding globulin (CBG) is a steroid binding protein that binds and transports cortisol in the blood circulation and is a potential target for endocrine disruption. An imbalance of cortisol in the body leads to many health problems. Induced fit docking of nine important and environmentally relevant phthalate plasticizers (DMP, BBP, DBP, DIBP, DnHP, DEHP, DINP, DnOP, DIDP) showed interactions with 10-19 amino acid residues of CBG. Comparison of the interacting residues of CBG with phthalate ligands and cortisol showed an overlapping of the majority (53-82%) of residues for each phthalate. Five of nine phthalate compounds and cortisol shared a hydrogen bonding interaction with the Arg-252 residue of CBG. Long-chain phthalates, such as DEHP, DINP, DnOP and DIDP displayed a higher binding affinity and formed a number of interactions with CBG in comparison to short-chain phthalates. The similarity in structural binding characteristics of phthalate compounds and native ligand cortisol suggested potential competitive conflicts in CBG-cortisol binding function and possible disruption of cortisol and progesterone homeostasis. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor.

    Science.gov (United States)

    Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J H; Risseeuw, Martijn D P; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

    2014-12-01

    Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Theory of substrate, Zeeman, and electron-phonon interaction effects on the quantum capacitance in graphene

    KAUST Repository

    Tahir, M.; Sabeeh, K.; Schwingenschlö gl, Udo; Shaukat, A.

    2013-01-01

    Since the discovery of graphene, a lot of interest has been attracted by the zeroth Landau level, which has no analog in the conventional two dimensional electron gas. Recently, lifting of the spin and valley degeneracies has been confirmed experimentally by capacitance measurements, while in transport experiments, this is difficult due to the scattering in the device. In this context, we model interaction effects on the quantum capacitance of graphene in the presence of a perpendicular magnetic field, finding good agreement with experiments. We demonstrate that the valley degeneracy is lifted by the substrate and by Kekule distortion, whereas the spin degeneracy is lifted by Zeeman interaction. The two cases can be distinguished by capacitance measurements.

  14. Theory of substrate, Zeeman, and electron-phonon interaction effects on the quantum capacitance in graphene

    KAUST Repository

    Tahir, M.

    2013-12-10

    Since the discovery of graphene, a lot of interest has been attracted by the zeroth Landau level, which has no analog in the conventional two dimensional electron gas. Recently, lifting of the spin and valley degeneracies has been confirmed experimentally by capacitance measurements, while in transport experiments, this is difficult due to the scattering in the device. In this context, we model interaction effects on the quantum capacitance of graphene in the presence of a perpendicular magnetic field, finding good agreement with experiments. We demonstrate that the valley degeneracy is lifted by the substrate and by Kekule distortion, whereas the spin degeneracy is lifted by Zeeman interaction. The two cases can be distinguished by capacitance measurements.

  15. Theory of substrate, Zeeman, and electron-phonon interaction effects on the quantum capacitance in graphene

    International Nuclear Information System (INIS)

    Tahir, M.; Sabeeh, K.; Shaukat, A.; Schwingenschlögl, U.

    2013-01-01

    Since the discovery of graphene, a lot of interest has been attracted by the zeroth Landau level, which has no analog in the conventional two dimensional electron gas. Recently, lifting of the spin and valley degeneracies has been confirmed experimentally by capacitance measurements, while in transport experiments, this is difficult due to the scattering in the device. In this context, we model interaction effects on the quantum capacitance of graphene in the presence of a perpendicular magnetic field, finding good agreement with experiments. We demonstrate that the valley degeneracy is lifted by the substrate and by Kekule distortion, whereas the spin degeneracy is lifted by Zeeman interaction. The two cases can be distinguished by capacitance measurements

  16. Identification of regions involved in substrate binding and dimer stabilization within the central domains of yeast Hsp40 Sis1.

    Directory of Open Access Journals (Sweden)

    Júlio C Borges

    Full Text Available Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1 Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2 The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3 The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4 Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

  17. Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

    Science.gov (United States)

    Nieto, Lidia; Tharun, Inga M; Balk, Mark; Wienk, Hans; Boelens, Rolf; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

    2015-11-20

    The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

  18. DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

    Science.gov (United States)

    Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

    2016-11-02

    Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

    Science.gov (United States)

    Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

    2014-03-07

    The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

  20. Direct determination of monolayer MoS2 and WSe2 exciton binding energies on insulating and metallic substrates

    KAUST Repository

    Park, Soohyung; Mutz, Niklas; Schultz, Thorsten; Blumstengel, Sylke; Han, Ali; Aljarb, Areej; Li, Lain-Jong; List-Kratochvil, Emil J W; Amsalem, Patrick; Koch, Norbert

    2018-01-01

    Understanding the excitonic nature of excited states in two-dimensional (2D) transition-metal dichalcogenides (TMDCs) is of key importance to make use of their optical and charge transport properties in optoelectronic applications. We contribute to this by the direct experimental determination of the exciton binding energy (E b,exc) of monolayer MoS2 and WSe2 on two fundamentally different substrates, i.e. the insulator sapphire and the metal gold. By combining angle-resolved direct and inverse photoelectron spectroscopy we measure the electronic band gap (E g), and by reflectance measurements the optical excitonic band gap (E exc). The difference of these two energies is E b,exc. The values of E g and E b,exc are 2.11 eV and 240 meV for MoS2 on sapphire, and 1.89 eV and 240 meV for WSe2 on sapphire. On Au E b,exc is decreased to 90 meV and 140 meV for MoS2 and WSe2, respectively. The significant E b,exc reduction is primarily due to a reduction of E g resulting from enhanced screening by the metal, while E exc is barely decreased for the metal support. Energy level diagrams determined at the K-point of the 2D TMDCs Brillouin zone show that MoS2 has more p-type character on Au as compared to sapphire, while WSe2 appears close to intrinsic on both. These results demonstrate that the impact of the dielectric environment of 2D TMDCs is more pronounced for individual charge carriers than for a correlated electron–hole pair, i.e. the exciton. A proper dielectric surrounding design for such 2D semiconductors can therefore be used to facilitate superior optoelectronic device function.

  1. Direct determination of monolayer MoS2 and WSe2 exciton binding energies on insulating and metallic substrates

    KAUST Repository

    Park, Soohyung

    2018-01-03

    Understanding the excitonic nature of excited states in two-dimensional (2D) transition-metal dichalcogenides (TMDCs) is of key importance to make use of their optical and charge transport properties in optoelectronic applications. We contribute to this by the direct experimental determination of the exciton binding energy (E b,exc) of monolayer MoS2 and WSe2 on two fundamentally different substrates, i.e. the insulator sapphire and the metal gold. By combining angle-resolved direct and inverse photoelectron spectroscopy we measure the electronic band gap (E g), and by reflectance measurements the optical excitonic band gap (E exc). The difference of these two energies is E b,exc. The values of E g and E b,exc are 2.11 eV and 240 meV for MoS2 on sapphire, and 1.89 eV and 240 meV for WSe2 on sapphire. On Au E b,exc is decreased to 90 meV and 140 meV for MoS2 and WSe2, respectively. The significant E b,exc reduction is primarily due to a reduction of E g resulting from enhanced screening by the metal, while E exc is barely decreased for the metal support. Energy level diagrams determined at the K-point of the 2D TMDCs Brillouin zone show that MoS2 has more p-type character on Au as compared to sapphire, while WSe2 appears close to intrinsic on both. These results demonstrate that the impact of the dielectric environment of 2D TMDCs is more pronounced for individual charge carriers than for a correlated electron–hole pair, i.e. the exciton. A proper dielectric surrounding design for such 2D semiconductors can therefore be used to facilitate superior optoelectronic device function.

  2. Direct determination of monolayer MoS2 and WSe2 exciton binding energies on insulating and metallic substrates

    Science.gov (United States)

    Park, Soohyung; Mutz, Niklas; Schultz, Thorsten; Blumstengel, Sylke; Han, Ali; Aljarb, Areej; Li, Lain-Jong; List-Kratochvil, Emil J. W.; Amsalem, Patrick; Koch, Norbert

    2018-04-01

    Understanding the excitonic nature of excited states in two-dimensional (2D) transition-metal dichalcogenides (TMDCs) is of key importance to make use of their optical and charge transport properties in optoelectronic applications. We contribute to this by the direct experimental determination of the exciton binding energy (E b,exc) of monolayer MoS2 and WSe2 on two fundamentally different substrates, i.e. the insulator sapphire and the metal gold. By combining angle-resolved direct and inverse photoelectron spectroscopy we measure the electronic band gap (E g), and by reflectance measurements the optical excitonic band gap (E exc). The difference of these two energies is E b,exc. The values of E g and E b,exc are 2.11 eV and 240 meV for MoS2 on sapphire, and 1.89 eV and 240 meV for WSe2 on sapphire. On Au E b,exc is decreased to 90 meV and 140 meV for MoS2 and WSe2, respectively. The significant E b,exc reduction is primarily due to a reduction of E g resulting from enhanced screening by the metal, while E exc is barely decreased for the metal support. Energy level diagrams determined at the K-point of the 2D TMDCs Brillouin zone show that MoS2 has more p-type character on Au as compared to sapphire, while WSe2 appears close to intrinsic on both. These results demonstrate that the impact of the dielectric environment of 2D TMDCs is more pronounced for individual charge carriers than for a correlated electron-hole pair, i.e. the exciton. A proper dielectric surrounding design for such 2D semiconductors can therefore be used to facilitate superior optoelectronic device function.

  3. Structural insight into the binding interactions of modeled structure of Arabidopsis thaliana urease with urea: an in silico study.

    Science.gov (United States)

    Yata, Vinod Kumar; Thapa, Arun; Mattaparthi, Venkata Satish Kumar

    2015-01-01

    Urease (EC 3.5.1.5., urea amidohydrolase) catalyzes the hydrolysis of urea to ammonia and carbon dioxide. Urease is present to a greater abundance in plants and plays significant role related to nitrogen recycling from urea. But little is known about the structure and function of the urease derived from the Arabidopsis thaliana, the model system of choice for research in plant biology. In this study, a three-dimensional structural model of A. thaliana urease was constructed using computer-aided molecular modeling technique. The characteristic structural features of the modeled structure were then studied using atomistic molecular dynamics simulation. It was observed that the modeled structure was stable and regions between residues index (50-80, 500-700) to be significantly flexible. From the docking studies, we detected the possible binding interactions of modeled urease with urea. Ala399, Ile675, Thr398, and Thr679 residues of A. thaliana urease were observed to be significantly involved in binding with the substrate urea. We also compared the docking studies of ureases from other sources such as Canavalia ensiformis, Helicobacter pylori, and Bacillus pasteurii. In addition, we carried out mutation analysis to find the highly mutable amino acid residues of modeled A. thaliana urease. In this particular study, we observed Met485, Tyr510, Ser786, Val426, and Lys765 to be highly mutable amino acids. These results are significant for the mutagenesis analysis. As a whole, this study expounds the salient structural features as well the binding interactions of the modeled structure of A. thaliana urease.

  4. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  5. Contribution of charge symmetry breaking interactions in binding energy difference of mirror nuclei

    International Nuclear Information System (INIS)

    Asghari, M.

    2006-01-01

    Nolen-Schiffer Anomaly in mirror nuclei due to the NN interactions with isospin mixing between T=0 and T=1 mesons of the same spin and parity are investigated. With the computation of coulomb energy along with the charge symmetry breaking effects provide a reasonably accurate description of binding energy differences between 39 Ca- 39 K , 41 Sc- 41 Ca mirror nuclei

  6. Binding behaviors of greenly synthesized silver nanoparticles - Lysozyme interaction: Spectroscopic approach

    Science.gov (United States)

    Roy, Swarup

    2018-02-01

    Interaction of greenly synthesized silver nanoparticles (SNP) and lysozyme (Lys) has been studied using spectroscopy. From UV-Vis study it is observed that a moderate association constant (Kapp) of 5.36 × 104 L/mol giving an indication of interaction. Fluorescence emission and time resolved study, confirm static mode of quenching phenomena and the binding constant (Kb) was 25.12, 3.98 and 1.99 × 103 L/mol at 298, 305 and 312 K respectively and the number of binding sites (n) was found to be ∼1. Using temperature dependent fluorimetric data, thermodynamic parameters calculated (Enthalpy change, ΔH = -143.95 kJ/mol, Entropy change, ΔS = -400.32 J/mol/K, Gibbs free energy change, ΔG = -24.66 kJ/mol at 298 K) and resulting insight indicative of weak force (van der Walls interaction & H-bonding) as key feature for the Lys-SNP interaction. By following Förster's non-radiative energy transfer (FRET) theory, average binding distance (r = 3.05 nm) was calculated and observed that nonradiative type energy transfer between SNP and Lys. What is more, circular dichroism (CD) spectra indicates presence of SNP does not display substantial alteration in the secondary structure of Lys. Hence, this results may be very useful for the well thought of essential aspects of binding between the Lys and SNP.

  7. RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Clémentine Delan-Forino

    2017-07-01

    Full Text Available Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly

  8. The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

    Science.gov (United States)

    Horton, R W; Lowther, S; Chivers, J; Jenner, P; Marsden, C D; Testa, B

    1988-08-01

    1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites. 8. Clebopride and Delagrange 2674 are structurally dissimilar to other BDZ ligands and represent another chemical structure to probe brain BDZ binding sites.

  9. Elucidation of relaxin-3 binding interactions in the extracellular loops of RXFP3

    Directory of Open Access Journals (Sweden)

    Ross eBathgate

    2013-02-01

    Full Text Available Relaxin-3 is a highly conserved neuropeptide in vertebrate species and binds to the Class A G protein-coupled receptor RXFP3. Relaxin-3 is involved in a wide range of behaviours, including feeding, stress responses, arousal and cognitive processes and therefore targeting of RXFP3 may be relevant for a range of neurological diseases. Structural knowledge of RXFP3 and its interaction with relaxin-3 would both increase our understanding of ligand recognition in GPCRs that respond to protein ligands and enable acceleration of the design of drug leads. In this study we have used comparative sequence analysis, molecular modelling and receptor mutagenesis to investigate the binding site of the native ligand human relaxin-3 (H3 relaxin on the human RXFP3 receptor. Previous structure function studies have demonstrated that arginine residues in the H3 relaxin B-chain are critical for binding interactions with the receptor extracellular loops and/or N-terminal domain. Hence we have concentrated on determining the ligand interacting sites in these domains and have focussed on glutamic (E and aspartic acid (D residues in these regions that may form electrostatic interactions with these critical arginine residues. Conserved D/E residues identified from vertebrate species multiple sequence alignments were mutated to Ala in human RXFP3 to test the effect of loss of amino acid side chain on receptor binding using both Eu-labelled relaxin-3 agonist. Finally data from mutagenesis experiments have been used in ligand docking simulations to a homology model of human RXFP3 based on the peptide-bound CXCR4 structure. These studies have resulted in a model of the relaxin-3 interaction with RXFP3 which will inform further interrogation of the agonist binding site.

  10. Interaction of the N-(3-Methylpyridin-2-ylamide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode.

    Directory of Open Access Journals (Sweden)

    Jessica Karlsson

    Full Text Available Combined fatty acid amide hydrolase (FAAH and cyclooxygenase (COX inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH(T488A-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R- and (S-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively, whereas the (S-enantiomer of Ibu-AM5 (IC50 0.59 μM was more potent than the (R-enantiomer (IC50 5.7 μM. Multiple inhibition experiments indicated that both (R-Flu-AM1 and (S-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.

  11. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    Science.gov (United States)

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

  12. Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions.

    Science.gov (United States)

    Su, Min-Gang; Weng, Julia Tzu-Ya; Hsu, Justin Bo-Kai; Huang, Kai-Yao; Chi, Yu-Hsiang; Lee, Tzong-Yi

    2017-12-21

    Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. The improved throughput of mass spectrometry (MS) or MS/MS technology has not only brought about a surge in proteome-scale studies, but also contributed to a fruitful list of identified PTMs. However, with the increase in the number of identified PTMs, perhaps the more crucial question is what kind of biological mechanisms these PTMs are involved in. This is particularly important in light of the fact that most protein-based pharmaceuticals deliver their therapeutic effects through some form of PTM. Yet, our understanding is still limited with respect to the local effects and frequency of PTM sites near pharmaceutical binding sites and the interfaces of protein-protein interaction (PPI). Understanding PTM's function is critical to our ability to manipulate the biological mechanisms of protein. In this study, to understand the regulation of protein functions by PTMs, we mapped 25,835 PTM sites to proteins with available three-dimensional (3D) structural information in the Protein Data Bank (PDB), including 1785 modified PTM sites on the 3D structure. Based on the acquired structural PTM sites, we proposed to use five properties for the structural characterization of PTM substrate sites: the spatial composition of amino acids, residues and side-chain orientations surrounding the PTM substrate sites, as well as the secondary structure, division of acidity and alkaline residues, and solvent-accessible surface area. We further mapped the structural PTM sites to the structures of drug binding and PPI sites, identifying a total of 1917 PTM sites that may affect PPI and 3951 PTM sites associated with drug-target binding. An integrated analytical platform (CruxPTM), with a variety of methods and online molecular docking

  13. Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB).

    Science.gov (United States)

    van Loon, Barbara; Samson, Leona D

    2013-03-01

    Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair. Copyright © 2012. Published by Elsevier B.V.

  14. Van der Waals interactions between planar substrate and tubular lipid membranes undergoing pearling instability

    Science.gov (United States)

    Valchev, G. S.; Djondjorov, P. A.; Vassilev, V. M.; Dantchev, D. M.

    2017-10-01

    In the current article we study the behavior of the van der Waals force between a planar substrate and an axisymmetric bilayer lipid membrane undergoing pearling instability, caused by uniform hydrostatic pressure difference. To do so, the recently suggested "surface integration approach" is used, which can be considered a generalization of the well known and widely used Derjaguin approximation. The static equilibrium shape after the occurrence of the instability is described in the framework of Helfrich's spontaneous curvature model. Some specific classes of exact analytical solutions to the corresponding shape equation are considered, and the components of the respective position vectors given in terms of elliptic integrals and Jacobi elliptic functions. The mutual orientation between the interacting objects is chosen such that the axis of revolution of the distorted cylinder be parallel to the plane bounding the substrate. Based on the discussed models and approaches we made some estimations for the studied force in real experimentally realizable systems, thus showing the possibility of pearling as an useful technique for reduction of the adhesion in variety of industrial processes using lipid membranes as carriers.

  15. Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

    Science.gov (United States)

    Herbst, R; Munemitsu, S; Ullrich, A

    1995-01-19

    The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

  16. Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

    Science.gov (United States)

    Diaz, Alejandra R; Core, Leighton J; Jiang, Min; Morelli, Michela; Chiang, Christina H; Szurmant, Hendrik; Perego, Marta

    2012-03-01

    Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

  17. Towards Automated Binding Affinity Prediction Using an Iterative Linear Interaction Energy Approach

    Directory of Open Access Journals (Sweden)

    C. Ruben Vosmeer

    2014-01-01

    Full Text Available Binding affinity prediction of potential drugs to target and off-target proteins is an essential asset in drug development. These predictions require the calculation of binding free energies. In such calculations, it is a major challenge to properly account for both the dynamic nature of the protein and the possible variety of ligand-binding orientations, while keeping computational costs tractable. Recently, an iterative Linear Interaction Energy (LIE approach was introduced, in which results from multiple simulations of a protein-ligand complex are combined into a single binding free energy using a Boltzmann weighting-based scheme. This method was shown to reach experimental accuracy for flexible proteins while retaining the computational efficiency of the general LIE approach. Here, we show that the iterative LIE approach can be used to predict binding affinities in an automated way. A workflow was designed using preselected protein conformations, automated ligand docking and clustering, and a (semi-automated molecular dynamics simulation setup. We show that using this workflow, binding affinities of aryloxypropanolamines to the malleable Cytochrome P450 2D6 enzyme can be predicted without a priori knowledge of dominant protein-ligand conformations. In addition, we provide an outlook for an approach to assess the quality of the LIE predictions, based on simulation outcomes only.

  18. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity......-contributing interactions are attributed to different domains and known to occur in two steps. Here, knowledge on chemokine and receptor domains involved in the first binding-step and the second activation-step is reviewed. A mechanism comprising at least two steps seems consistent; however, several intermediate...... interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly...

  19. Binding interaction of atorvastatin with bovine serum albumin: Spectroscopic methods and molecular docking

    Science.gov (United States)

    Wang, Qi; Huang, Chuan-ren; Jiang, Min; Zhu, Ying-yao; Wang, Jing; Chen, Jun; Shi, Jie-hua

    2016-03-01

    The interaction of atorvastatin with bovine serum albumin (BSA) was investigated using multi-spectroscopic methods and molecular docking technique for providing important insight into further elucidating the store and transport process of atorvastatin in the body and the mechanism of action and pharmacokinetics. The experimental results revealed that the fluorescence quenching mechanism of BSA induced atorvastatin was a combined dynamic and static quenching. The binding constant and number of binding site of atorvastatin with BSA under simulated physiological conditions (pH = 7.4) were 1.41 × 105 M- 1 and about 1 at 310 K, respectively. The values of the enthalpic change (ΔH0), entropic change (ΔS0) and Gibbs free energy (ΔG0) in the binding process of atorvastatin with BSA at 310 K were negative, suggesting that the binding process of atorvastatin and BSA was spontaneous and the main interaction forces were van der Waals force and hydrogen bonding interaction. Moreover, atorvastatin was bound into the subdomain IIA (site I) of BSA, resulting in a slight change of the conformation of BSA.

  20. Interactions of Poly(amidoamine) Dendrimers with Human Serum Albumin: Binding Constants and Mechanisms

    OpenAIRE

    Giri, Jyotsnendu; Diallo, Mamadou S.; Simpson, André J.; Liu, Yi; Goddard, William A., III; Kumar, Rajeev; Woods, Gwen C.

    2011-01-01

    The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K_b) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To g...

  1. The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

    OpenAIRE

    Horton, R. W.; Lowther, S.; Chivers, J.; Jenner, P.; Marsden, C. D.; Testa, B.

    1988-01-01

    1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiap...

  2. Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

    Science.gov (United States)

    Zhu, Yunye; Huang, Shengxiong; Miao, Min; Tang, Xiaofeng; Yue, Junyang; Wang, Wenjie; Liu, Yongsheng

    2015-06-01

    One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitation assays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

  3. Active site CP-loop dynamics modulate substrate binding, catalysis, oligomerization, stability, over-oxidation and recycling of 2-Cys Peroxiredoxins.

    Science.gov (United States)

    Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

    2018-04-01

    Peroxiredoxins (Prxs) catalyse the rapid reduction of hydrogen peroxide, organic hydroperoxide and peroxynitrite, using a fully conserved peroxidatic cysteine (C P ) located in a conserved sequence Pxxx(T/S)xxC P motif known as C P -loop. In addition, Prxs are involved in cellular signaling pathways and regulate several redox-dependent process related disease. The effective catalysis of Prxs is associated with alterations in the C P -loop between reduced, Fully Folded (FF), and oxidized, Locally Unfolded (LU) conformations, which are linked to dramatic changes in the oligomeric structure. Despite many studies, little is known about the precise structural and dynamic roles of the C P -loop on Prxs functions. Herein, the comprehensive biochemical and biophysical studies on Escherichia coli alkyl hydroperoxide reductase subunit C (EcAhpC) and the C P -loop mutants, EcAhpC-F45A and EcAhpC-F45P reveal that the reduced form of the C P -loop adopts conformational dynamics, which is essential for effective peroxide reduction. Furthermore, the point mutants alter the structure and dynamics of the reduced form of the C P -loop and, thereby, affect substrate binding, catalysis, oligomerization, stability and overoxidiation. In the oxidized form, due to restricted C P -loop dynamics, the EcAhpC-F45P mutant favours a decamer formation, which enhances the effective recycling by physiological reductases compared to wild-type EcAhpC. In addition, the study reveals that residue F45 increases the specificity of Prxs-reductase interactions. Based on these studies, we propose an evolution of the C P -loop with confined sequence conservation within Prxs subfamilies that might optimize the functional adaptation of Prxs into various physiological roles. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  5. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    Science.gov (United States)

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  6. Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III).

    Science.gov (United States)

    Lesner, A; Brzozowski, K; Kupryszewski, G; Rolka, K

    2000-03-05

    A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme. Copyright 2000 Academic Press.

  7. Large-dimension configuration-interaction calculations of positron binding to the group-II atoms

    International Nuclear Information System (INIS)

    Bromley, M. W. J.; Mitroy, J.

    2006-01-01

    The configuration-interaction (CI) method is applied to the calculation of the structures of a number of positron binding systems, including e + Be, e + Mg, e + Ca, and e + Sr. These calculations were carried out in orbital spaces containing about 200 electron and 200 positron orbitals up to l=12. Despite the very large dimensions, the binding energy and annihilation rate converge slowly with l, and the final values do contain an appreciable correction obtained by extrapolating the calculation to the l→∞ limit. The binding energies were 0.00317 hartree for e + Be, 0.0170 hartree for e + Mg, 0.0189 hartree for e + Ca, and 0.0131 hartree for e + Sr

  8. DIBS: a repository of disordered binding sites mediating interactions with ordered proteins.

    Science.gov (United States)

    Schad, Eva; Fichó, Erzsébet; Pancsa, Rita; Simon, István; Dosztányi, Zsuzsanna; Mészáros, Bálint

    2018-02-01

    Intrinsically Disordered Proteins (IDPs) mediate crucial protein-protein interactions, most notably in signaling and regulation. As their importance is increasingly recognized, the detailed analyses of specific IDP interactions opened up new opportunities for therapeutic targeting. Yet, large scale information about IDP-mediated interactions in structural and functional details are lacking, hindering the understanding of the mechanisms underlying this distinct binding mode. Here, we present DIBS, the first comprehensive, curated collection of complexes between IDPs and ordered proteins. DIBS not only describes by far the highest number of cases, it also provides the dissociation constants of their interactions, as well as the description of potential post-translational modifications modulating the binding strength and linear motifs involved in the binding. Together with the wide range of structural and functional annotations, DIBS will provide the cornerstone for structural and functional studies of IDP complexes. DIBS is freely accessible at http://dibs.enzim.ttk.mta.hu/. The DIBS application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. dosztanyi@caesar.elte.hu or bmeszaros@caesar.elte.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

  9. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yezhong [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China); College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China); Zhou Bo [College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China); Zhang Xiaoping; Huang Ping [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China); Li Chaohong [Education Ministry Key Laboratory for Oral Biomedical Engineering, School of Stomatology, Wuhan University, Wuhan 430072 (China); Liu Yi [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China) and College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China)], E-mail: prof.liuyi@263.net

    2009-04-30

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K{sub a}) between MG and BSA at four different temperatures were obtained to be 3.734 x 10{sup 4}, 3.264 x 10{sup 4}, 2.718 x 10{sup 4}, and 2.164 x 10{sup 4} L mol{sup -1}, respectively. The enthalpy change ({delta}H) and entropy change ({delta}S) were calculated to be -27.25 kJ mol{sup -1} and -11.23 J mol{sup -1} K{sup -1}, indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the {alpha}-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.

  10. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    International Nuclear Information System (INIS)

    Zhang Yezhong; Zhou Bo; Zhang Xiaoping; Huang Ping; Li Chaohong; Liu Yi

    2009-01-01

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K a ) between MG and BSA at four different temperatures were obtained to be 3.734 x 10 4 , 3.264 x 10 4 , 2.718 x 10 4 , and 2.164 x 10 4 L mol -1 , respectively. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -27.25 kJ mol -1 and -11.23 J mol -1 K -1 , indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the α-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules

  11. Structural analysis of protein-ligand interactions: the binding of endogenous compounds and of synthetic drugs.

    Science.gov (United States)

    Gallina, Anna M; Bork, Peer; Bordo, Domenico

    2014-02-01

    The large number of macromolecular structures deposited with the Protein Data Bank (PDB) describing complexes between proteins and either physiological compounds or synthetic drugs made it possible a systematic analysis of the interactions occurring between proteins and their ligands. In this work, the binding pockets of about 4000 PDB protein-ligand complexes were investigated and amino acid and interaction types were analyzed. The residues observed with lowest frequency in protein sequences, Trp, His, Met, Tyr, and Phe, turned out to be the most abundant in binding pockets. Significant differences between drug-like and physiological compounds were found. On average, physiological compounds establish with respect to drugs about twice as many hydrogen bonds with protein atoms, whereas drugs rely more on hydrophobic interactions to establish target selectivity. The large number of PDB structures describing homologous proteins in complex with the same ligand made it possible to analyze the conservation of binding pocket residues among homologous protein structures bound to the same ligand, showing that Gly, Glu, Arg, Asp, His, and Thr are more conserved than other amino acids. Also in the cases in which the same ligand is bound to unrelated proteins, the binding pockets showed significant conservation in the residue types. In this case, the probability of co-occurrence of the same amino acid type in the binding pockets could be up to thirteen times higher than that expected on a random basis. The trends identified in this study may provide an useful guideline in the process of drug design and lead optimization. Copyright © 2014 John Wiley & Sons, Ltd.

  12. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  13. A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2015-01-01

    Full Text Available Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD of heat shock 70 kDa protein (PDB: 1HJO with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD simulation. Human DNA binding domain of p53 motif (SCMGGMNR retrieved from UniProt (UniProtKB: P04637 was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

  14. Effect of van der Waals interactions on the structural and binding properties of GaSe

    Energy Technology Data Exchange (ETDEWEB)

    Sarkisov, Sergey Y., E-mail: sarkisov@mail.tsu.ru [Tomsk State University, Lenin Avenue 36, 634050 Tomsk (Russian Federation); Kosobutsky, Alexey V., E-mail: kosobutsky@kemsu.ru [Tomsk State University, Lenin Avenue 36, 634050 Tomsk (Russian Federation); Kemerovo State University, Krasnaya 6, 650043 Kemerovo (Russian Federation); Shandakov, Sergey D. [Kemerovo State University, Krasnaya 6, 650043 Kemerovo (Russian Federation)

    2015-12-15

    The influence of van der Waals interactions on the lattice parameters, band structure, elastic moduli and binding energy of layered GaSe compound has been studied using projector-augmented wave method within density functional theory. We employed the conventional local/semilocal exchange-correlation functionals and recently developed van der Waals functionals which are able to describe dispersion forces. It is found that application of van der Waals density functionals allows to substantially increase the accuracy of calculations of the lattice constants a and c and interlayer distance in GaSe at ambient conditions and under hydrostatic pressure. The pressure dependences of the a-parameter, Ga–Ga, Ga–Se bond lengths and Ga–Ga–Se bond angle are characterized by a relatively low curvature, while c(p) has a distinct downward bowing due to nonlinear shrinking of the interlayer spacing. From the calculated binding energy curves we deduce the interlayer binding energy of GaSe, which is found to be in the range 0.172–0.197 eV/layer (14.2–16.2 meV/Å{sup 2}). - Highlights: • Effects of van der Waals interactions are analyzed using advanced density functionals. • Calculations with vdW-corrected functionals closely agree with experiment. • Interlayer binding energy of GaSe is estimated to be 14.2–16.2 meV/Å{sup 2}.

  15. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  16. Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

    Science.gov (United States)

    Makarska-Bialokoz, Magdalena

    2017-05-01

    The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

  17. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  18. Neural substrates underlying the tendency to accept anger-infused ultimatum offers during dynamic social interactions.

    Science.gov (United States)

    Gilam, Gadi; Lin, Tamar; Raz, Gal; Azrielant, Shir; Fruchter, Eyal; Ariely, Dan; Hendler, Talma

    2015-10-15

    In managing our way through interpersonal conflict, anger might be crucial in determining whether the dispute escalates to aggressive behaviors or resolves cooperatively. The Ultimatum Game (UG) is a social decision-making paradigm that provides a framework for studying interpersonal conflict over division of monetary resources. Unfair monetary UG-offers elicit anger and while accepting them engages regulatory processes, rejecting them is regarded as an aggressive retribution. Ventro-medial prefrontal-cortex (vmPFC) activity has been shown to relate to idiosyncratic tendencies in accepting unfair offers possibly through its role in emotion regulation. Nevertheless, standard UG paradigms lack fundamental aspects of real-life social interactions in which one reacts to other people in a response contingent fashion. To uncover the neural substrates underlying the tendency to accept anger-infused ultimatum offers during dynamic social interactions, we incorporated on-line verbal negotiations with an obnoxious partner in a repeated-UG during fMRI scanning. We hypothesized that vmPFC activity will differentiate between individuals with high or low monetary gains accumulated throughout the game and reflect a divergence in the associated emotional experience. We found that as individuals gained more money, they reported less anger but also more positive feelings and had slower sympathetic response. In addition, high-gain individuals had increased vmPFC activity, but also decreased brainstem activity, which possibly reflected the locus coeruleus. During the more angering unfair offers, these individuals had increased dorsal-posterior Insula (dpI) activity which functionally coupled to the medial-thalamus (mT). Finally, both vmPFC activity and dpI-mT connectivity contributed to increased gain, possibly by modulating the ongoing subjective emotional experience. These ecologically valid findings point towards a neural mechanism that might nurture pro-social interactions by

  19. The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma.

    Science.gov (United States)

    Maranchie, Jodi K; Vasselli, James R; Riss, Joseph; Bonifacino, Juan S; Linehan, W Marston; Klausner, Richard D

    2002-04-01

    Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

  20. Origin of low sodium capacity in graphite and generally weak substrate binding of Na and Mg among alkali and alkaline earth metals.

    Science.gov (United States)

    Liu, Yuanyue; Merinov, Boris V; Goddard, William A

    2016-04-05

    It is well known that graphite has a low capacity for Na but a high capacity for other alkali metals. The growing interest in alternative cation batteries beyond Li makes it particularly important to elucidate the origin of this behavior, which is not well understood. In examining this question, we find a quite general phenomenon: among the alkali and alkaline earth metals, Na and Mg generally have the weakest chemical binding to a given substrate, compared with the other elements in the same column of the periodic table. We demonstrate this with quantum mechanics calculations for a wide range of substrate materials (not limited to C) covering a variety of structures and chemical compositions. The phenomenon arises from the competition between trends in the ionization energy and the ion-substrate coupling, down the columns of the periodic table. Consequently, the cathodic voltage for Na and Mg is expected to be lower than those for other metals in the same column. This generality provides a basis for analyzing the binding of alkali and alkaline earth metal atoms over a broad range of systems.

  1. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch

    2015-01-01

    ) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced...... to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4...

  2. Binding free energy calculations to rationalize the interactions of huprines with acetylcholinesterase.

    Science.gov (United States)

    Nascimento, Érica C M; Oliva, Mónica; Andrés, Juan

    2018-05-01

    In the present study, the binding free energy of a family of huprines with acetylcholinesterase (AChE) is calculated by means of the free energy perturbation method, based on hybrid quantum mechanics and molecular mechanics potentials. Binding free energy calculations and the analysis of the geometrical parameters highlight the importance of the stereochemistry of huprines in AChE inhibition. Binding isotope effects are calculated to unravel the interactions between ligands and the gorge of AChE. New chemical insights are provided to explain and rationalize the experimental results. A good correlation with the experimental data is found for a family of inhibitors with moderate differences in the enzyme affinity. The analysis of the geometrical parameters and interaction energy per residue reveals that Asp72, Glu199, and His440 contribute significantly to the network of interactions between active site residues, which stabilize the inhibitors in the gorge. It seems that a cooperative effect of the residues of the gorge determines the affinity of the enzyme for these inhibitors, where Asp72, Glu199, and His440 make a prominent contribution.

  3. Binding free energy calculations to rationalize the interactions of huprines with acetylcholinesterase

    Science.gov (United States)

    Nascimento, Érica C. M.; Oliva, Mónica; Andrés, Juan

    2018-05-01

    In the present study, the binding free energy of a family of huprines with acetylcholinesterase (AChE) is calculated by means of the free energy perturbation method, based on hybrid quantum mechanics and molecular mechanics potentials. Binding free energy calculations and the analysis of the geometrical parameters highlight the importance of the stereochemistry of huprines in AChE inhibition. Binding isotope effects are calculated to unravel the interactions between ligands and the gorge of AChE. New chemical insights are provided to explain and rationalize the experimental results. A good correlation with the experimental data is found for a family of inhibitors with moderate differences in the enzyme affinity. The analysis of the geometrical parameters and interaction energy per residue reveals that Asp72, Glu199, and His440 contribute significantly to the network of interactions between active site residues, which stabilize the inhibitors in the gorge. It seems that a cooperative effect of the residues of the gorge determines the affinity of the enzyme for these inhibitors, where Asp72, Glu199, and His440 make a prominent contribution.

  4. The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions.

    Science.gov (United States)

    Beckmann, Benedikt M; Castello, Alfredo; Medenbach, Jan

    2016-06-01

    Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.

  5. Effects of trace of nitrogen on the helium atmospheric pressure plasma jet interacting with a dielectric substrate

    Science.gov (United States)

    Ning, Wenjun; Dai, Dong; Zhang, Yuhui; Han, Yongxia; Li, Licheng

    2018-03-01

    Experimental observations and simulation results regarding a pure He atmospheric pressure plasma jet (APPJ) and He  +  N2 APPJs interacting with a downstream dielectric substrate are presented in this paper. Experiments utilizing spatial-temporal imaging show that, in the case of the pure He APPJ, an annular plasma-substrate interaction pattern is formed. With the introduction of N2, the plasma is more uniformly distributed on the substrate surface, appearing a solid interaction pattern. The experimental measurements indicate 0.5% N2 mixture is the optimal condition to achieve the most intense discharge, while the plasma-substrate contact area is slightly reduced by 6.1% in comparison to that of the pure He APPJ. A 2D self-consistent fluid model is constructed to provide insights into the role of the addition of trace of N2 on the discharge dynamics. The discharge morphologies predicated by the model is in principle consistent with the experimental observations. The simulation reveals that the conversion from the annular plasma-substrate interaction pattern to the solid one is attributed to the synthetic effect of the addition of N2 and the presentence of the substrate acting as the cathode to enhance the local electric field. In the solid interaction pattern, the Penning ionization makes a significant contribution to the surface discharge, especially in the afterglow region. The dominant positive ions (N2+ and N4+ ) and the reactive oxygen and nitrogen species including O and N gain remarkable increment in the flux intensity to the central surface, which merits great application potential.

  6. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    Science.gov (United States)

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

    Science.gov (United States)

    Dayan, Avraham; Babin, Gilad; Ganoth, Assaf; Kayouf, Nivin Samir; Nitoker Eliaz, Neta; Mukkala, Srijana; Tsfadia, Yossi; Fleminger, Gideon

    2017-08-01

    Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO 2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO 2 -binding capabilities with TiBP. Intrigued by the unique TiO 2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO 2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO 2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO 2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO 2 through similar regions located far from the active site and the dimerization sites. The putative TiO 2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Interaction of silicene with β-Si3N4(0001)/Si(111) substrate; energetics and electronic properties

    International Nuclear Information System (INIS)

    Filippone, Francesco

    2014-01-01

    The free-standing, quasi-2D layer of Si is known as silicene, in analogy with graphene. Much effort is devoted in the study of silicene, since, similarly to graphene, it shows a very high electron mobility. The interaction of silicene with a hybrid substrate, β-Si 3 N 4 (0001)/Si(111), exposing the β-Si 3 N 4 (0001) surface, has been studied by means of Density Functional calculations, with van der Waals interactions included. Once deepened the most important structural and electronic features of the hybrid substrate, we demonstrated that an electron transfer occurs from the substrate to the silicene layer. In turn, such an electron transfer can be modulated by the doping of the substrate. The β-Si 3 N 4 /silicene interaction appears to be strong enough to ensure adequate adsorption stability. It is also shown that electronic states of substrate and adsorbate still remain decoupled, paving the way for the exploitation of the peculiar electron mobility properties of the silicene layer. A detailed analysis in both direct and reciprocal space is reported. (paper)

  9. Participation of Glutamate-354 of the CP43 Polypeptide in the Ligation of Mn and the Binding of Substrate Water in Photosystem II

    Energy Technology Data Exchange (ETDEWEB)

    Service, Rachel; Yano, Junko; McConnell, Iain; Hwang, Hong Jin; Niks, Dimitri; Hille, Russ; Wydrzynski, Tom; Burnap, Robert; Hillier, Warwick; Debus, Richard

    2010-09-30

    . The EPR and FTIR data implied that 76 -82 percent of CP43-E354Q PSII centers can achieve the S2 state and that most of these can achieve the S3 state, but no evidence for advancement beyond the S3 state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn4Ca cluster in the S2 state, the FTIR and H218O exchange data show that the mutation strongly influences other properties of the Mn4Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S1 to S2 transition and weakening the binding of both substrate water molecules (or water derived ligands), especially the one that exchanges rapidly in the S3 state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn4Ca cluster in the S1 state as a bridging ligand between two metal ions, but provide no compelling evidence that this residue changes its coordination mode during the S1 to S2 transition. The H218O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S3 state.

  10. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-01-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  11. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  12. Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

    Directory of Open Access Journals (Sweden)

    Michelle P Christie

    Full Text Available The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

  13. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

  14. Prediction of vitamin interacting residues in a vitamin binding protein using evolutionary information.

    Science.gov (United States)

    Panwar, Bharat; Gupta, Sudheer; Raghava, Gajendra P S

    2013-02-07

    The vitamins are important cofactors in various enzymatic-reactions. In past, many inhibitors have been designed against vitamin binding pockets in order to inhibit vitamin-protein interactions. Thus, it is important to identify vitamin interacting residues in a protein. It is possible to detect vitamin-binding pockets on a protein, if its tertiary structure is known. Unfortunately tertiary structures of limited proteins are available. Therefore, it is important to develop in-silico models for predicting vitamin interacting residues in protein from its primary structure. In this study, first we compared protein-interacting residues of vitamins with other ligands using Two Sample Logo (TSL). It was observed that ATP, GTP, NAD, FAD and mannose preferred {G,R,K,S,H}, {G,K,T,S,D,N}, {T,G,Y}, {G,Y,W} and {Y,D,W,N,E} residues respectively, whereas vitamins preferred {Y,F,S,W,T,G,H} residues for the interaction with proteins. Furthermore, compositional information of preferred and non-preferred residues along with patterns-specificity was also observed within different vitamin-classes. Vitamins A, B and B6 preferred {F,I,W,Y,L,V}, {S,Y,G,T,H,W,N,E} and {S,T,G,H,Y,N} interacting residues respectively. It suggested that protein-binding patterns of vitamins are different from other ligands, and motivated us to develop separate predictor for vitamins and their sub-classes. The four different prediction modules, (i) vitamin interacting residues (VIRs), (ii) vitamin-A interacting residues (VAIRs), (iii) vitamin-B interacting residues (VBIRs) and (iv) pyridoxal-5-phosphate (vitamin B6) interacting residues (PLPIRs) have been developed. We applied various classifiers of SVM, BayesNet, NaiveBayes, ComplementNaiveBayes, NaiveBayesMultinomial, RandomForest and IBk etc., as machine learning techniques, using binary and Position-Specific Scoring Matrix (PSSM) features of protein sequences. Finally, we selected best performing SVM modules and obtained highest MCC of 0.53, 0.48, 0.61, 0

  15. Ag films deposited on Si and Ti: How the film-substrate interaction influences the nanoscale film morphology

    Science.gov (United States)

    Ruffino, F.; Torrisi, V.

    2017-11-01

    Submicron-thick Ag films were sputter deposited, at room temperature, on Si, covered by the native SiO2 layer, and on Ti, covered by the native TiO2 layer, under normal and oblique deposition angle. The aim of this work was to study the morphological differences in the grown Ag films on the two substrates when fixed all the other deposition parameters. In fact, the surface diffusivity of the Ag adatoms is different on the two substrates (higher on the SiO2 surface) due to the different Ag-SiO2 and Ag-TiO2 atomic interactions. So, the effect of the adatoms surface diffusivity, as determined by the adatoms-substrate interaction, on the final film morphology was analyzed. To this end, microscopic analyses were used to study the morphology of the grown Ag films. Even if the homologous temperature prescribes that the Ag film grows on both substrates in the zone I described by the structure zone model some significant differences are observed on the basis of the supporting substrate. In the normal incidence condition, on the SiO2/Si surface a dense close-packed Ag film exhibiting a smooth surface is obtained, while on the TiO2/Ti surface a more columnar film morphology is formed. In the oblique incidence condition the columnar morphology for the Ag film occurs both on SiO2/Si and TiO2/Ti but a higher porous columnar film is obtained on TiO2/Ti due to the lower Ag diffusivity. These results indicate that the adatoms diffusivity on the substrate as determined by the adatom-surface interaction (in addition to the substrate temperature) strongly determines the final film nanostructure.

  16. [Binding interaction of harpagoside and bovine serum albumin: spectroscopic methodologies and molecular docking].

    Science.gov (United States)

    Cao, Tuan-Wu; Huang, Wen-Bing; Shi, Jian-Wei; He, Wei

    2018-03-01

    Scrophularia ningpoensis has exhibited a variety of biological activities and been used as a pharmaceutical product for the treatment of inflammatory ailment, rheumatoid arthritis, osteoarthritis and so on. Harpagoside (HAR) is considerer as a main bioactive compound in this plant. Serum albumin has important physiological roles in transportation, distribution and metabolism of many endogenous and exogenous substances in body. It is of great significance to study the interaction mechanism between HAR and bovine serum albumin (BSA). The mechanism of interaction between HAR and BSA was investigated using 2D and 3D fluorescence, synchronous florescence, ultraviolet spectroscopy and molecular docking. According to the analysis of fluorescence spectra, HAR could strongly quench the fluorescence of BSA, and the static quenching process indicated that the decrease in the quenching constant was observed with the increase in temperature. The magnitude of binding constants (KA) was more than 1×10⁵ L·mol⁻¹, and the number of binding sites(n) was approximate to 1. The thermodynamic parameters were calculated through analysis of fluorescence data with Stern-Volmer and Van't Hoff equation. The calculated enthalpy change (ΔH) and entropy change (ΔS) implied that the main interaction forces of HAR with BSA were the bonding interaction between van der Waals forces and hydrogen. The negative values of energy (ΔG) demonstrated that the binding of HAR with BSA was a spontaneous and exothermic process. The binding distance(r) between HAR and BSA was calculated to be about 2.80 nm based on the theory of Frster's non-radiation energy transfer, which indicated that energy is likely to be transfer from BSA to HAR. Both synchronous and 3D florescence spectroscopy clearly revealed that the microenvironment and conformation of BSA changed during the binding interaction between HAR and BSA. The molecular docking analysis revealed HAR is more inclined to BSA and human serum albumin

  17. Interaction Entropy: A New Paradigm for Highly Efficient and Reliable Computation of Protein-Ligand Binding Free Energy.

    Science.gov (United States)

    Duan, Lili; Liu, Xiao; Zhang, John Z H

    2016-05-04

    Efficient and reliable calculation of protein-ligand binding free energy is a grand challenge in computational biology and is of critical importance in drug design and many other molecular recognition problems. The main challenge lies in the calculation of entropic contribution to protein-ligand binding or interaction systems. In this report, we present a new interaction entropy method which is theoretically rigorous, computationally efficient, and numerically reliable for calculating entropic contribution to free energy in protein-ligand binding and other interaction processes. Drastically different from the widely employed but extremely expensive normal mode method for calculating entropy change in protein-ligand binding, the new method calculates the entropic component (interaction entropy or -TΔS) of the binding free energy directly from molecular dynamics simulation without any extra computational cost. Extensive study of over a dozen randomly selected protein-ligand binding systems demonstrated that this interaction entropy method is both computationally efficient and numerically reliable and is vastly superior to the standard normal mode approach. This interaction entropy paradigm introduces a novel and intuitive conceptual understanding of the entropic effect in protein-ligand binding and other general interaction systems as well as a practical method for highly efficient calculation of this effect.

  18. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    Science.gov (United States)

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

  19. Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

    International Nuclear Information System (INIS)

    Warner, Dennis R.; Bhattacherjee, Vasker; Yin, Xiaolong; Singh, Saurabh; Mukhopadhyay, Partha; Pisano, M. Michele; Greene, Robert M.

    2004-01-01

    The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue

  20. Crystal complexes of a predicted S-adenosylmethionine-dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain

    Energy Technology Data Exchange (ETDEWEB)

    Miller, D.J.; Ouellette, N.; Evodokimova, E.; Savchenko, A.; Edwards, A.; Anderson, W.F. (Toronto); (NWU)

    2010-03-08

    S-adenosyl-L-methionine-dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans. The genes for many of the bacterial homologs are located within operons involved in cell wall synthesis and cell division. Despite preliminary biochemical studies in E. coli and B. subtilis, the substrate specificity of this group of more than 150 proteins is unknown. As part of the Midwest Center for Structural Genomics initiative (www.mcsg.anl.gov), we have determined the structure of TM0872 in complexes with AdoMet and with S-adenosyl-L-homocysteine (AdoHcy). As predicted, TM0872 has a typical MT domain, and binds endogenous AdoMet, or co-crystallized AdoHcy, in a manner consistent with other known MT structures. In addition, TM0872 has a second domain that is novel among MTs in both its location in the sequence and its structure. The second domain likely acts in substrate recognition and binding, and there is a potential substrate-binding cleft spanning the two domains. This long and narrow cleft is lined with positively charged residues which are located opposite the S{sup +}-CH{sub 3} bond, suggesting that a negatively charged molecule might be targeted for catalysis. However, AdoMet and AdoHcy are both buried, and access to the methyl group would presumably require structural rearrangement. These TM0872 crystal structures offer the first structural glimpses at this phylogenetically conserved sequence family.

  1. Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.

    Science.gov (United States)

    Skagseth, Susann; Christopeit, Tony; Akhter, Sundus; Bayer, Annette; Samuelsen, Ørjan; Leiros, Hanna-Kirsti S

    2017-08-01

    Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC 50 ) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC 50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant ( K D ) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than l-captopril (IC 50 = 47 μM; K D = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril. Copyright

  2. Interactions of Cu-substrates with titanium-alloyed Sn-Zn solders

    Directory of Open Access Journals (Sweden)

    Soares D.

    2006-01-01

    Full Text Available The interactions of copper substrate with titanium-alloyed Sn-Zn eutectic solders have been studied. Two series of experiments have been performed. The first one consisted in differential thermal analyses of Sn-Zn nearly eutectic alloys containing from 1.3 to 2.2 wt. % Ti. Diffusion couples consisted of Cu-wires and Sn-Zn-Ti liquid solders, produced at 250 and 275 OC have been prepared in the second series,. The contact times were up to 3600 s. The contact zones have been characterized by optical and scanning electron microscope. Two layers have been found along the interfaces solid/liquid. The first and the second layers are identical, respectively, with γ and ε phases of the Cu-Zn system. No changes of the chemical compositions were detected for the tested temperatures and reaction times. Continuous parabolic growth of the total diffusion zone thickness with the time of diffusion is observed. The growth is due mainly to one the formed layers (γ while the thickness of the ε-phase layer, stays almost constant for all tested diffusion times and temperatures.

  3. Dynamic, adaptive changes in MAO-A binding after alterations in substrate availability: an in vivo [11C]-harmine positron emission tomography study

    Science.gov (United States)

    Sacher, Julia; Rabiner, Eugenii A; Clark, Michael; Rusjan, Pablo; Soliman, Alexandra; Boskovic, Rada; Kish, Stephen J; Wilson, Alan A; Houle, Sylvain; Meyer, Jeffrey H

    2012-01-01

    Monoamine oxidase A (MAO-A) is an important target in the pathophysiology and therapeutics of major depressive disorder, aggression, and neurodegenerative conditions. We measured the effect of changes in MAO-A substrate on MAO-A binding in regions implicated in affective and neurodegenerative disease with [11C]-harmine positron emission tomography in healthy volunteers. Monoamine oxidase A VT, an index of MAO-A density, was decreased (mean: 14%±9%) following tryptophan depletion in prefrontal cortex (PMAO-A in maintaining monoamine neurotransmitter homeostasis by rapidly compensating fluctuating monoamine levels. PMID:22186668

  4. Effects of Mutations in the Substrate-Binding Domain of Poly[(R)-3-Hydroxybutyrate] (PHB) Depolymerase from Ralstonia pickettii T1 on PHB Degradation▿

    OpenAIRE

    Hiraishi, Tomohiro; Hirahara, Yoko; Doi, Yoshiharu; Maeda, Mizuo; Taguchi, Seiichi

    2006-01-01

    Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZRpiT1) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZRpiT1 was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZRpiT1. Genetic...

  5. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    International Nuclear Information System (INIS)

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

    2006-01-01

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

  6. Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation.

    Science.gov (United States)

    Afdhal, N H; Ostrow, J D; Koehler, R; Niu, N; Groen, A K; Veis, A; Nunes, D P; Offner, G D

    1995-11-01

    Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

  7. Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

    Science.gov (United States)

    Kamina, Anyango D; Williams, Noreen

    2017-01-01

    RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

  8. Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

    Science.gov (United States)

    Henschler, R; Rüster, B; Steimle, A; Hansmann, H L; Walker, W; Montag, T; Seifried, E

    2005-08-01

    Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

  9. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

    Directory of Open Access Journals (Sweden)

    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  10. Hydrophobic interaction chromatography in dual salt system increases protein binding capacity.

    Science.gov (United States)

    Senczuk, Anna M; Klinke, Ralph; Arakawa, Tsutomu; Vedantham, Ganesh; Yigzaw, Yinges

    2009-08-01

    Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

  11. Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

    Science.gov (United States)

    Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

    2018-04-30

    Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

    International Nuclear Information System (INIS)

    Ahluwalia, Unnati; Deep, Shashank

    2012-01-01

    Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

  13. Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

    NARCIS (Netherlands)

    Schmidt, Marco F; Groves, Matthew R; Rademann, Jörg

    2011-01-01

    Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic

  14. Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

    Science.gov (United States)

    Denys, A; Allain, F; Carpentier, M; Spik, G

    1998-12-15

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

  15. Odorant binding protein 69a connects social interaction to modulation of social responsiveness in Drosophila.

    Science.gov (United States)

    Bentzur, Assa; Shmueli, Anat; Omesi, Liora; Ryvkin, Julia; Knapp, Jon-Michael; Parnas, Moshe; Davis, Fred P; Shohat-Ophir, Galit

    2018-04-01

    Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In Drosophila melanogaster, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. Although it is the most studied pheromone in flies, the mechanisms that determine the complexity of the response, its intensity and final output with respect to social context, sex and prior interaction, are still not well understood. Here we explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. Odorant binding protein 69a (Obp69a) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. We show that activation of cVA-sensing neurons is sufficeint to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies.

  16. Interacting proteins on human spermatozoa: adaptive evolution of the binding of semenogelin I to EPPIN.

    Directory of Open Access Journals (Sweden)

    Erick J R Silva

    Full Text Available Semenogelin I (SEMG1 is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins.

  17. Snake cytotoxins bind to membranes via interactions with phosphatidylserine head groups of lipids.

    Directory of Open Access Journals (Sweden)

    Anastasia G Konshina

    Full Text Available The major representatives of Elapidae snake venom, cytotoxins (CTs, share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS, and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against

  18. Catalyst–substrate interaction and growth delay in vapor–liquid–solid nanowire growth

    Science.gov (United States)

    Kolíbal, Miroslav; Pejchal, Tomáš; Musálek, Tomáš; Šikola, Tomáš

    2018-05-01

    Understanding of the initial stage of nanowire growth on a bulk substrate is crucial for the rational design of nanowire building blocks in future electronic and optoelectronic devices. Here, we provide in situ scanning electron microscopy and Auger microscopy analysis of the initial stage of Au-catalyzed Ge nanowire growth on different substrates. Real-time microscopy imaging and elementally resolved spectroscopy clearly show that the catalyst dissolves the underlying substrate if held above a certain temperature. If the substrate dissolution is blocked (or in the case of heteroepitaxy) the catalyst needs to be filled with nanowire material from the external supply, which significantly increases the initial growth delay. The experiments presented here reveal the important role of the substrate in metal-catalyzed nanowire growth and pave the way for different growth delay mitigation strategies.

  19. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    Science.gov (United States)

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  20. Physical adsorption at the nanoscale: Towards controllable scaling of the substrate-adsorbate van der Waals interaction

    Science.gov (United States)

    Ambrosetti, Alberto; Silvestrelli, Pier Luigi; Tkatchenko, Alexandre

    2017-06-01

    The Lifshitz-Zaremba-Kohn (LZK) theory is commonly considered as the correct large-distance limit for the van der Waals (vdW) interaction of adsorbates (atoms, molecules, or nanoparticles) with solid substrates. In the standard approximate form, implicitly based on local dielectric functions, the LZK approach predicts universal power laws for vdW interactions depending only on the dimensionality of the interacting objects. However, recent experimental findings are challenging the universality of this theoretical approach at finite distances of relevance for nanoscale assembly. Here, we present a combined analytical and numerical many-body study demonstrating that physical adsorption can be significantly enhanced at the nanoscale. Regardless of the band gap or the nature of the adsorbate specie, we find deviations from conventional LZK power laws that extend to separation distances of up to 10-20 nm. Comparison with recent experimental observations of ultra-long-ranged vdW interactions in the delamination of graphene from a silicon substrate reveals qualitative agreement with the present theory. The sensitivity of vdW interactions to the substrate response and to the adsorbate characteristic excitation frequency also suggests that adsorption strength can be effectively tuned in experiments, paving the way to an improved control of physical adsorption at the nanoscale.

  1. The localization of key Bacillus subtilis penicillin binding proteins during cell growth is determined by substrate availability

    NARCIS (Netherlands)

    Lages, Marta Carolina Afonso; Beilharz, Katrin; Angeles, Danae Morales; Veening, Jan-Willem; Scheffers, Dirk-Jan

    2013-01-01

    The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan (PG) that is synthesized by penicillin binding proteins (PBPs). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for

  2. Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues

    International Nuclear Information System (INIS)

    Tanner, N.K.; Hanna, M.M.; Abelson, J.

    1988-01-01

    Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs

  3. Interaction between cardiac myosin-binding protein C and formin Fhod3.

    Science.gov (United States)

    Matsuyama, Sho; Kage, Yohko; Fujimoto, Noriko; Ushijima, Tomoki; Tsuruda, Toshihiro; Kitamura, Kazuo; Shiose, Akira; Asada, Yujiro; Sumimoto, Hideki; Takeya, Ryu

    2018-05-08

    Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction.

  4. Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

    OpenAIRE

    Magdalena Opalińska; Katarzyna Parys; Hanna Jańska

    2017-01-01

    Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we...

  5. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Fish, A.; Rucktooa, P.; Khruschov, A.Y.; Osipov, A.V.; Ziganshin, R.H.; D'Hoedt, D.; Bertrand, D.; Sixma, T.K.; Smit, A.B.; Tsetlin, V.I.

    2009-01-01

    α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7

  6. The Effect of Distal Interactions on O2-Binding to Heme

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Dasmeh, Pouria

    2013-01-01

    This paper reports DFT-computed electronic ground states, Mössbauer isomer shifts, O-O and Fe-O vibration frequencies, and thermodynamics of O2-binding of heme models representing different distal (position E7) interactions, strictly validated against experimental data. Based on the results...... hydrogen bonds can further reduce isomer shifts up to 0.07 mm/s. The O-O stretch vibration, the O-O distance, and the isomer shift possess substantial heuristic value in interpreting electronic structure, whereas other properties are less effective, based on computed correlation coefficients. Shorter Fe...

  7. Label-free detection of biomolecular interaction — DNA — Antimicrobial peptide binding

    DEFF Research Database (Denmark)

    Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

    2011-01-01

    the molecule. In particular, surface plasmon resonance (SPR) sensors have been already demonstrated suitable for food-safety control, label-free screening for various disease markers in bodily fluids, as well as for real-time continuous monitoring of drug levels in intensive care environment. We envisage...... of plasmon based biosensors to the study of the interaction of Antimicrobial peptide IL4 and DNA. Our results indicate high affinity binding between IL4 and DNA thereby preventing DNA replication and eventually killing the affected cell. We speculate that this is common for a large class of Antimicrobial...

  8. Weakly hydrated surfaces and the binding interactions of small biological solutes.

    Science.gov (United States)

    Brady, John W; Tavagnacco, Letizia; Ehrlich, Laurent; Chen, Mo; Schnupf, Udo; Himmel, Michael E; Saboungi, Marie-Louise; Cesàro, Attilio

    2012-04-01

    Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

  9. Interaction between cellular retinoic acid-binding protein II and histone hypoacetylation in renal cell carcinoma

    OpenAIRE

    Viroj Wiwanitkit

    2008-01-01

    Renal cell carcinoma is a rare but serious malignancy. Since a reduction in the level of retinoic acid receptor beta 2 (RARbeta2) expression in cancer cells due in part to histone hypoacetylation which is controlled by histone deacetylase (HD), the study on the interaction between cellular retinoic acid-binding proteins II (CRABP II), which is proposed to have its potential influence on retinoic acid (RA) response, and HD can be useful. Comparing to CARBP II and HD, the CARBP II-HD poses the ...

  10. Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

    Science.gov (United States)

    Hoggett, J G; Kellett, G L

    1976-06-15

    A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.

  11. Interactions between Cellulolytic Enzymes with Native, Autohydrolysis, and Technical Lignins and the Effect of a Polysorbate Amphiphile in Reducing Nonproductive Binding.

    Science.gov (United States)

    Fritz, Consuelo; Ferrer, Ana; Salas, Carlos; Jameel, Hasan; Rojas, Orlando J

    2015-12-14

    Understanding enzyme-substrate interactions is critical in designing strategies for bioconversion of lignocellulosic biomass. In this study we monitored molecular events, in situ and in real time, including the adsorption and desorption of cellulolytic enzymes on lignins and cellulose, by using quartz crystal microgravimetry and surface plasmon resonance. The effect of a nonionic surface active molecule was also elucidated. Three lignin substrates relevant to the sugar platform in biorefinery efforts were considered, namely, hardwood autohydrolysis cellulolytic (HWAH), hardwood native cellulolytic (MPCEL), and nonwood native cellulolytic (WSCEL) lignin. In addition, Kraft lignins derived from softwoods (SWK) and hardwoods (HWK) were used as references. The results indicated a high affinity between the lignins with both, monocomponent and multicomponent enzymes. More importantly, the addition of nonionic surfactants at concentrations above their critical micelle concentration reduced remarkably (by over 90%) the nonproductive interactions between the cellulolytic enzymes and the lignins. This effect was hypothesized to be a consequence of the balance of hydrophobic and hydrogen bonding interactions. Moreover, the reduction of surface roughness and increased wettability of lignin surfaces upon surfactant treatment contributed to a lower affinity with the enzymes. Conformational changes of cellulases were observed upon their adsorption on lignin carrying preadsorbed surfactant. Weak electrostatic interactions were determined in aqueous media at pH between 4.8 and 5.5 for the native cellulolytic lignins (MPCEL and WSCEL), whereby a ∼20% reduction in the enzyme affinity was observed. This was mainly explained by electrostatic interactions (osmotic pressure effects) between charged lignins and cellulases. Noteworthy, adsorption of nonionic surfactants onto cellulose, in the form cellulose nanofibrils, did not affect its hydrolytic conversion. Overall, our results

  12. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

    Science.gov (United States)

    Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

  13. Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

    OpenAIRE

    Denys, A; Allain, F; Carpentier, M; Spik, G

    1998-01-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes...

  14. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

    Science.gov (United States)

    Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

    2002-06-28

    Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

  15. Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

    OpenAIRE

    Stephanie J. Ruiz; Gea K. Schuurman-Wolters; Bert Poolman

    2016-01-01

    BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC) importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related co...

  16. Predicting Ligand Binding Sites on Protein Surfaces by 3-Dimensional Probability Density Distributions of Interacting Atoms

    Science.gov (United States)

    Jian, Jhih-Wei; Elumalai, Pavadai; Pitti, Thejkiran; Wu, Chih Yuan; Tsai, Keng-Chang; Chang, Jeng-Yih; Peng, Hung-Pin; Yang, An-Suei

    2016-01-01

    Predicting ligand binding sites (LBSs) on protein structures, which are obtained either from experimental or computational methods, is a useful first step in functional annotation or structure-based drug design for the protein structures. In this work, the structure-based machine learning algorithm ISMBLab-LIG was developed to predict LBSs on protein surfaces with input attributes derived from the three-dimensional probability density maps of interacting atoms, which were reconstructed on the query protein surfaces and were relatively insensitive to local conformational variations of the tentative ligand binding sites. The prediction accuracy of the ISMBLab-LIG predictors is comparable to that of the best LBS predictors benchmarked on several well-established testing datasets. More importantly, the ISMBLab-LIG algorithm has substantial tolerance to the prediction uncertainties of computationally derived protein structure models. As such, the method is particularly useful for predicting LBSs not only on experimental protein structures without known LBS templates in the database but also on computationally predicted model protein structures with structural uncertainties in the tentative ligand binding sites. PMID:27513851

  17. Interaction of metallic nanoparticles with dielectric substrates: effect of optical constants

    International Nuclear Information System (INIS)

    Hutter, Tanya; Elliott, Stephen R; Mahajan, Sumeet

    2013-01-01

    In this paper, we study the local-field enhancement in a system of a metallic nanoparticle placed very near to a dielectric substrate. In such systems, intense electric fields are localized in the gap between the particle and the substrate, creating a ‘hot-spot’ under appropriate excitation conditions. We use finite-element numerical simulations in order to study the field enhancement in this dielectric–metal system. More specifically, we show how the optical properties of the dielectric substrate (n and k) affect the plasmonic field enhancement in the nano-gap. We also analyze the degree of field confinement in the gap and discuss it in the context of utilization for surface-enhanced Raman scattering. We finally show the fields generated by real substrates and compare them to metallic ones. (paper)

  18. Simple Ligand–Receptor Interaction Descriptor (SILIRID for alignment-free binding site comparison

    Directory of Open Access Journals (Sweden)

    Vladimir Chupakhin

    2014-06-01

    Full Text Available We describe SILIRID (Simple Ligand–Receptor Interaction Descriptor, a novel fixed size descriptor characterizing protein–ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion. Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein–ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91. SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM: sc-PDB SILIRID data form clusters corresponding to different protein types.

  19. Simple Ligand-Receptor Interaction Descriptor (SILIRID) for alignment-free binding site comparison.

    Science.gov (United States)

    Chupakhin, Vladimir; Marcou, Gilles; Gaspar, Helena; Varnek, Alexandre

    2014-06-01

    We describe SILIRID (Simple Ligand-Receptor Interaction Descriptor), a novel fixed size descriptor characterizing protein-ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs) by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor) and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion). Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein-ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91). SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM): sc-PDB SILIRID data form clusters corresponding to different protein types.

  20. Partial genetic deletion of neuregulin 1 and adolescent stress interact to alter NMDA receptor binding in the medial prefrontal cortex

    Directory of Open Access Journals (Sweden)

    Tariq Waseem Chohan

    2014-09-01

    Full Text Available Schizophrenia is thought to arise due to a complex interaction between genetic and environmental factors during early neurodevelopment. We have recently shown that partial genetic deletion of the schizophrenia susceptibility gene neuregulin 1 (Nrg1 and adolescent stress interact to disturb sensorimotor gating, neuroendocrine activity and dendritic morphology in mice. Both stress and Nrg1 may have converging effects upon N-methyl-D-aspartate receptors (NMDARs which are implicated in the pathogenesis of schizophrenia, sensorimotor gating and dendritic spine plasticity. Using an identical repeated restraint stress paradigm to our previous study, here we determined NMDAR binding across various brain regions in adolescent Nrg1 heterozygous (HET and wild-type (WT mice using [3H] MK-801 autoradiography. Repeated restraint stress increased NMDAR binding in the ventral part of the lateral septum (LSV and the dentate gyrus (DG of the hippocampus irrespective of genotype. Partial genetic deletion of Nrg1 interacted with adolescent stress to promote an altered pattern of NMDAR binding in the infralimbic (IL subregion of the medial prefrontal cortex. In the IL, whilst stress tended to increase NMDAR binding in WT mice, it decreased binding in Nrg1 HET mice. However in the DG, stress selectively increased the expression of NMDAR binding in Nrg1 HET mice but not WT mice. These results demonstrate a Nrg1-stress interaction during adolescence on NMDAR binding in the medial prefrontal cortex.

  1. Darapladib Binds to Lipoprotein-Associated Phospholipase A2 with Meaningful Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Do, Kyungrok; Chang, Byungha; Shin, Jae Min; No, Kyoung Tai; Lee, Jeeyoung [Bioinformatics and Molecular Design Research Center, Seoul (Korea, Republic of); Kim, Chul; Yea, Sangjun; Song, Miyoung [Korea Institute of Oriental Medicine, Daejeon (Korea, Republic of)

    2014-01-15

    Lipoprotein-associated phospholipase A{sub 2} (Lp-PLA{sub 2}) is a crucial enzyme in atherosclerosis as a potential drug target. The most remarkable Lp-PLA{sub 2} inhibitory drug is Darapladib. We determined the binding pose of Darapladib to Lp-PLA{sub 2} through docking study. Darapladib formed two hydrogen bonding interactions with the side chain of Tyr160 and Gln352 and several pi-pi interactions with aromatic and aliphatic hydrophobic residues of Lp-PLA{sub 2}. It is known that the dietylpropan-amine moiety of Darapladib has influence on the improvement of its oral bioavailability and we supposed this in our docking results.

  2. Moiré superlattice-level stick-slip instability originated from geometrically corrugated graphene on a strongly interacting substrate

    Science.gov (United States)

    Shi, Ruoyu; Gao, Lei; Lu, Hongliang; Li, Qunyang; Ma, Tian-Bao; Guo, Hui; Du, Shixuan; Feng, Xi-Qiao; Zhang, Shuai; Liu, Yanmin; Cheng, Peng; Hu, Yuan-Zhong; Gao, Hong-Jun; Luo, Jianbin

    2017-06-01

    Two dimensional (2D) materials often exhibit novel properties due to various coupling effects with their supporting substrates. Here, using friction force microscopy (FFM), we report an unusual moiré superlattice-level stick-slip instability on monolayer graphene epitaxially grown on Ru(0 0 0 1) substrate. Instead of smooth friction modulation, a significant long-range stick-slip sawtooth modulation emerges with a period coinciding with the moiré superlattice structure, which is robust against high external loads and leads to an additional channel of energy dissipation. In contrast, the long-range stick-slip instability reduces to smooth friction modulation on graphene/Ir(1 1 1) substrate. The moiré superlattice-level slip instability could be attributed to the large sliding energy barrier, which arises from the morphological corrugation of graphene on Ru(0 0 0 1) surface as indicated by density functional theory (DFT) calculations. The locally steep humps acting as obstacles opposing the tip sliding, originates from the strong interfacial electronic interaction between graphene and Ru(0 0 0 1). This study opens an avenue for modulating friction by tuning the interfacial atomic interaction between 2D materials and their substrates.

  3. Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

    Science.gov (United States)

    Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

    1994-11-04

    Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

  4. Probing the interaction of brain fatty acid binding protein (B-FABP with model membranes.

    Directory of Open Access Journals (Sweden)

    Fábio Dyszy

    Full Text Available Brain fatty acid-binding protein (B-FABP interacts with biological membranes and delivers polyunsaturated fatty acids (FAs via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called "portal region", formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.

  5. Conglutinin binds the HIV-1 envelope glycoprotein gp 160 and inhibits its interaction with cell membrane CD4

    DEFF Research Database (Denmark)

    Andersen, Ove; Sørensen, A M; Svehag, S E

    1991-01-01

    The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose...... residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160...... of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein....

  6. Investigating the Turing conditions for diffusion-driven instability in the presence of a binding immobile substrate

    Czech Academy of Sciences Publication Activity Database

    Korvasová, K.; Gaffney, E. A.; Maini, P.K.; Ferreira, M.A.; Klika, Václav

    2015-01-01

    Roč. 367, February (2015), s. 286-295 ISSN 0022-5193 Institutional support: RVO:61388998 Keywords : turing instability * non-diffusive substrate * pattern formation Subject RIV: BJ - Thermodynamics Impact factor: 2.049, year: 2015 http://ac.els-cdn.com/S0022519314006766/1-s2.0-S0022519314006766-main.pdf?_tid=63ec0858-9ffa-11e5-969b-00000aacb35d&acdnat=1449833527_e470798087aa42f7ca3b2efcfffc48cf

  7. Beyond Ribosomal Binding: The Increased Polarity and Aberrant Molecular Interactions of 3-epi-deoxynivalenol

    Directory of Open Access Journals (Sweden)

    Yousef I. Hassan

    2016-09-01

    Full Text Available Deoxynivalenol (DON is a secondary fungal metabolite and contaminant mycotoxin that is widely detected in wheat and corn products cultivated around the world. Bio-remediation methods have been extensively studied in the past two decades and promising ways to reduce DON-associated toxicities have been reported. Bacterial epimerization of DON at the C3 carbon was recently reported to induce a significant loss in the bio-toxicity of the resulting stereoisomer (3-epi-DON in comparison to the parental compound, DON. In an earlier study, we confirmed the diminished bio-potency of 3-epi-DON using different mammalian cell lines and mouse models and mechanistically attributed it to the reduced binding of 3-epi-DON within the ribosomal peptidyl transferase center (PTC. In the current study and by inspecting the chromatographic behavior of 3-epi-DON and its molecular interactions with a well-characterized enzyme, Fusarium graminearum Tri101 acetyltransferase, we provide the evidence that the C3 carbon epimerization of DON influences its molecular interactions beyond the abrogated PTC binding.

  8. Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain.

    Directory of Open Access Journals (Sweden)

    Tingjun Hou

    2006-01-01

    Full Text Available Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3 domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well.

  9. Prediction of vitamin interacting residues in a vitamin binding protein using evolutionary information

    Directory of Open Access Journals (Sweden)

    Panwar Bharat

    2013-02-01

    Full Text Available Abstract Background The vitamins are important cofactors in various enzymatic-reactions. In past, many inhibitors have been designed against vitamin binding pockets in order to inhibit vitamin-protein interactions. Thus, it is important to identify vitamin interacting residues in a protein. It is possible to detect vitamin-binding pockets on a protein, if its tertiary structure is known. Unfortunately tertiary structures of limited proteins are available. Therefore, it is important to develop in-silico models for predicting vitamin interacting residues in protein from its primary structure. Results In this study, first we compared protein-interacting residues of vitamins with other ligands using Two Sample Logo (TSL. It was observed that ATP, GTP, NAD, FAD and mannose preferred {G,R,K,S,H}, {G,K,T,S,D,N}, {T,G,Y}, {G,Y,W} and {Y,D,W,N,E} residues respectively, whereas vitamins preferred {Y,F,S,W,T,G,H} residues for the interaction with proteins. Furthermore, compositional information of preferred and non-preferred residues along with patterns-specificity was also observed within different vitamin-classes. Vitamins A, B and B6 preferred {F,I,W,Y,L,V}, {S,Y,G,T,H,W,N,E} and {S,T,G,H,Y,N} interacting residues respectively. It suggested that protein-binding patterns of vitamins are different from other ligands, and motivated us to develop separate predictor for vitamins and their sub-classes. The four different prediction modules, (i vitamin interacting residues (VIRs, (ii vitamin-A interacting residues (VAIRs, (iii vitamin-B interacting residues (VBIRs and (iv pyridoxal-5-phosphate (vitamin B6 interacting residues (PLPIRs have been developed. We applied various classifiers of SVM, BayesNet, NaiveBayes, ComplementNaiveBayes, NaiveBayesMultinomial, RandomForest and IBk etc., as machine learning techniques, using binary and Position-Specific Scoring Matrix (PSSM features of protein sequences. Finally, we selected best performing SVM modules and

  10. Dielectric constant of graphene-on-polarized substrate: A tight ...

    Indian Academy of Sciences (India)

    2017-06-24

    Jun 24, 2017 ... We report here a microscopic tight-binding theoretical study of the dynamic dielectric response of graphene-on-polarizable substrate with impurity. The Hamiltonian consists of first, second and third nearest neighbour electron hopping interactions besides doping and substrate-induced effects on graphene.

  11. Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries

    International Nuclear Information System (INIS)

    Alvarez, P.J.J.; Vogel, T.M.

    1991-01-01

    Release of petroleum hydrocarbons in the environment is a widespread occurrence. One particular concern is the contamination of drinking water sources by the toxic, water-soluble, and mobile petroleum components benzene, toluene, and xylene (BTX). Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds

  12. Magnus-induced ratchet effects for skyrmions interacting with asymmetric substrates

    Science.gov (United States)

    Reichhardt, C.; Ray, D.; Olson Reichhardt, C. J.

    2015-07-01

    We show using numerical simulations that pronounced ratchet effects can occur for ac driven skyrmions moving over asymmetric quasi-one-dimensional substrates. We find a new type of ratchet effect called a Magnus-induced transverse ratchet that arises when the ac driving force is applied perpendicular rather than parallel to the asymmetry direction of the substrate. This transverse ratchet effect only occurs when the Magnus term is finite, and the threshold ac amplitude needed to induce it decreases as the Magnus term becomes more prominent. Ratcheting skyrmions follow ordered orbits in which the net displacement parallel to the substrate asymmetry direction is quantized. Skyrmion ratchets represent a new ac current-based method for controlling skyrmion positions and motion for spintronic applications.

  13. The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.

    Science.gov (United States)

    Lindfors, Hanna E; Drijfhout, Jan Wouter; Ubbink, Marcellus

    2012-06-01

    The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction. Copyright © 2012 Wiley Periodicals, Inc.

  14. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

    Science.gov (United States)

    Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

    2013-08-01

    Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

  15. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    Science.gov (United States)

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

  16. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.

    2002-01-01

    The fab1 mutant of Arabidopsis is partially deficient in activity of ß-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a c......DNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  17. Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains

    DEFF Research Database (Denmark)

    Haq, S Raza; Chi, Celestine N; Bach, Anders

    2012-01-01

    Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. We have here...... used short peptides as a model system for intrinsically disordered proteins. Linear free-energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate......-limiting barrier for binding, in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins towards their targets are generally...

  18. Involvement of a putative substrate binding site in the biogenesis and assembly of phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

    Science.gov (United States)

    Di Bartolomeo, Francesca; Doan, Kim Nguyen; Athenstaedt, Karin; Becker, Thomas; Daum, Günther

    2017-07-01

    In the yeast Saccharomyces cerevisiae, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) produces the largest amount of cellular phosphatidylethanolamine (PE). Psd1p is synthesized as a larger precursor on cytosolic ribosomes and then imported into mitochondria in a three-step processing event leading to the formation of an α-subunit and a β-subunit. The α-subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding. Here, we present a molecular analysis of this consensus motif for the function of Psd1p by using Psd1p variants bearing either deletions or point mutations in this region. Our data show that mutations in this motif affect processing and stability of Psd1p, and consequently the enzyme's activity. Thus, we conclude that this consensus motif is essential for structural integrity and processing of Psd1p. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

    Science.gov (United States)

    Sikorra, Stefan; Litschko, Christa; Müller, Carina; Thiel, Nadine; Galli, Thierry; Eichner, Timo; Binz, Thomas

    2016-01-29

    Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. The Effect of Substrate-Bulk Interaction on Hydrolysis Modeling in Anaerobic Digestion Process

    Directory of Open Access Journals (Sweden)

    Antonio Panico

    2014-11-01

    Full Text Available In an Anaerobic Digestion (AD process treating particulate substrates, the size of solids is expected to negatively affect the rate of hydrolysis step and consequently influence the performance of the whole process. To avoid any disadvantage due to size of solids, expensive pre-treatments aimed at disintegrating and solubilizing substrates are commonly conducted prior to AD. This practice is doubtlessly successful, but not always necessary, since some organic substrates, although particulate, once immersed in water, tend to solubilize immediately. This aspect, if properly considered, could result in saving money and time in the AD process, as well as refining the development and calibration of AD mathematical models. The present study is actually aimed at demonstrating, through experiments and mathematical simulations, different results deriving from the AD process performed, under the same operating conditions, on two different substrates, i.e. homemade pasta and carrot batons, having the same particle size, but different chemical composition and texture. Experimental outcomes highlighted the effect of particles size on bio-methane production only from the bio-methanation potential tests (BMP conducted on carrot batons. Similar results were obtained by mathematical model calibration, i.e., different kinetic constants for differently-sized carrot batons and same kinetic constant for differently-sized homemade pasta solids.

  1. Interactions between nitrate and chloride in nutrient solutions for substrate grown tomato

    NARCIS (Netherlands)

    Voogt, W.; Sonneveld, C.

    2004-01-01

    In two successive experiments tomato was grown at different Cl and NO3 concentrations in the root environment with rockwool as a sub-strate. The EC value in the nutrient solution was fairly constant, varying between 3.5 and 4.0 dS m-1 in all treatments. The NO3 concentrations in the treatments

  2. Interfacial Interaction of Oxidatively Cured Hydrogen Silsesquioxane Spin-On-Glass Enamel with Stainless Steel Substrate

    DEFF Research Database (Denmark)

    Lampert, Felix; Kadkhodazadeh, Shima; Jensen, Annemette H.

    2017-01-01

    interfacial duplex-oxide with an outer zone composed of Fe2O3 in a SiO2-x matrix and an inner zone composed of complex (Cr3+,Fe2+,Mn2+)-oxides. Moreover, a Cr depletion of the substrate in the immediate vicinity of the surface was observed. It was concluded that the interfacial formation is controlled...

  3. Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

    Science.gov (United States)

    Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

    2011-01-01

    HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

  4. Characterization of Glutaredoxin Fe-S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy.

    Science.gov (United States)

    Albetel, Angela-Nadia; Outten, Caryn E

    2018-01-01

    Monothiol glutaredoxins (Grxs) with a conserved Cys-Gly-Phe-Ser (CGFS) active site are iron-sulfur (Fe-S) cluster-binding proteins that interact with a variety of partner proteins and perform crucial roles in iron metabolism including Fe-S cluster transfer, Fe-S cluster repair, and iron signaling. Various analytical and spectroscopic methods are currently being used to monitor and characterize glutaredoxin Fe-S cluster-dependent interactions at the molecular level. The electronic, magnetic, and vibrational properties of the protein-bound Fe-S cluster provide a convenient handle to probe the structure, function, and coordination chemistry of Grx complexes. However, some limitations arise from sample preparation requirements, complexity of individual techniques, or the necessity for combining multiple methods in order to achieve a complete investigation. In this chapter, we focus on the use of UV-visible circular dichroism spectroscopy as a fast and simple initial approach for investigating glutaredoxin Fe-S cluster-dependent interactions. © 2018 Elsevier Inc. All rights reserved.

  5. Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

    DEFF Research Database (Denmark)

    Hachem, Maher Abou; Bozonnet, Sophie; Willemoës, Martin

    2006-01-01

    Barley a-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for ß-cyclodextrin by surface plasmon resonan...

  6. Interaction between cellular retinoic acid-binding protein II and histone hypoacetylation in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Viroj Wiwanitkit

    2008-04-01

    Full Text Available Renal cell carcinoma is a rare but serious malignancy. Since a reduction in the level of retinoic acid receptor beta 2 (RARbeta2 expression in cancer cells due in part to histone hypoacetylation which is controlled by histone deacetylase (HD, the study on the interaction between cellular retinoic acid-binding proteins II (CRABP II, which is proposed to have its potential influence on retinoic acid (RA response, and HD can be useful. Comparing to CARBP II and HD, the CARBP II-HD poses the same function and biological process as HD. This can confirm that HD has a significant suppressive effect on the expression of CARBP II. Therefore, reduction in the level of RARbeta2 expression in cancer cells can be expected and this can lead to failure in treatment of renal cell carcinoma with RA. The author hereby purpose that additional HD inhibitor should be added into the regiment of RA to increase the effectiveness of treatment.

  7. Sequence walkers: a graphical method to display how binding proteins interact with DNA or RNA sequences | Center for Cancer Research

    Science.gov (United States)

    A graphical method is presented for displaying how binding proteins and other macromolecules interact with individual bases of nucleotide sequences. Characters representing the sequence are either oriented normally and placed above a line indicating favorable contact, or upside-down and placed below the line indicating unfavorable contact. The positive or negative height of each letter shows the contribution of that base to the average sequence conservation of the binding site, as represented by a sequence logo.

  8. Characterizing the binding interaction between antimalarial artemether (AMT) and bovine serum albumin (BSA): Spectroscopic and molecular docking methods.

    Science.gov (United States)

    Shi, Jie-Hua; Pan, Dong-Qi; Wang, Xiou-Xiou; Liu, Ting-Ting; Jiang, Min; Wang, Qi

    2016-09-01

    Artemether (AMT), a peroxide sesquiterpenoides, has been widely used as an antimalarial for the treatment of multiple drug-resistant strains of plasmodium falciparum malaria. In this work, the binding interaction of AMT with bovine serum albumin (BSA) under the imitated physiological conditions (pH7.4) was investigated by UV spectroscopy, fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), three-dimensional fluorescence spectroscopy and molecular docking methods. The experimental results indicated that there was a change in UV absorption of BSA along with a slight red shift of absorption wavelength, indicating that the interaction of AMT with BSA occurred. The intrinsic fluorescence of BSA was quenched by AMT due to the formation of AMT-BSA complex. The number of binding sites (n) and binding constant of AMT-BSA complex were about 1 and 2.63×10(3)M(-1) at 298K, respectively, suggesting that there was stronger binding interaction of AMT with BSA. Based on the analysis of the signs and magnitudes of the free energy change (ΔG(0)), enthalpic change (ΔH(0)) and entropic change (ΔS(0)) in the binding process, it can be concluded that the binding of AMT with BSA was enthalpy-driven process due to |ΔH°|>|TΔS°|. The results of experiment and molecular docking confirmed the main interaction forces between AMT and BSA were van der Waals force. And, there was a slight change in the BSA conformation after binding AMT but BSA still retains its secondary structure α-helicity. However, it had been confirmed that AMT binds on the interface between sub-domain IIA and IIB of BSA. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Circular dichroism study of the interaction between mutagens and bilirubin bound to different binding sites of serum albumins

    Science.gov (United States)

    Orlov, Sergey; Goncharova, Iryna; Urbanová, Marie

    Although recent investigations have shown that bilirubin not only has a negative role in the organism but also exhibits significant antimutagenic properties, the mechanisms of interactions between bilirubin and mutagens are not clear. In this study, interaction between bilirubin bound to different binding sites of mammalian serum albumins with structural analogues of the mutagens 2-aminofluorene, 2,7-diaminofluorene and mutagen 2,4,7-trinitrofluorenone were investigated by circular dichroism and absorption spectroscopy. Homological human and bovine serum albumins were used as chiral matrices, which preferentially bind different conformers of bilirubin in the primary binding sites and make it observable by circular dichroism. These molecular systems approximated a real system for the study of mutagens in blood serum. Differences between the interaction of bilirubin bound to primary and to secondary binding sites of serum albumins with mutagens were shown. For bilirubin bound to secondary binding sites with low affinity, partial displacement and the formation of self-associates were observed in all studied mutagens. The associates of bilirubin bound to primary binding sites of serum albumins are formed with 2-aminofluorene and 2,4,7-trinitrofluorenone. It was proposed that 2,7-diaminofluorene does not interact with bilirubin bound to primary sites of human and bovine serum albumins due to the spatial hindrance of the albumins binding domains. The spatial arrangement of the bilirubin bound to serum albumin along with the studied mutagens was modelled using ligand docking, which revealed a possibility of an arrangement of the both bilirubin and 2-aminofluorene and 2,4,7-trinitrofluorenone in the primary binding site of human serum albumin.

  10. Interaction between LSD and dopamine D2/3 binding sites in pig brain.

    Science.gov (United States)

    Minuzzi, Luciano; Nomikos, George G; Wade, Mark R; Jensen, Svend B; Olsen, Aage K; Cumming, Paul

    2005-06-15

    The psychoactive properties of the hallucinogen LSD have frequently been attributed to high affinity interactions with serotonin 5HT2 receptors in brain. Possible effects of LSD on dopamine D2/3 receptor availability have not previously been investigated in living brain. Therefore, we used PET to map the binding potential (pB) of [11C]raclopride in brain of three pigs, first in a baseline condition, and again at 1 and 4 h after administration of LSD (2.5 microg/kg, i.v.). There was a progressive treatment effect in striatum, where the pB was significantly reduced by 19% at 4 h after LSD administration. Concomitant maps of cerebral blood flow did not reveal significant changes in perfusion during this interval. Subsequent in vitro studies showed that LSD displaced [3H]raclopride (2 nM) from pig brain cryostat sections with an IC50 of 275 nM according to a one-site model. Fitting of a two-site model to the data suggested the presence of a component of the displacement curves with a subnanomolar IC50, comprising 20% of the total [3H]raclopride binding. In microdialysis experiments, LSD at similar and higher doses did not evoke changes in the interstitial concentration of dopamine or its acidic metabolites in rat striatum. Together, these results are consistent with a direct interaction between LSD and a portion of dopamine D2/3 receptors in pig brain, possibly contributing to the psychopharmacology of LSD. (c) 2005 Wiley-Liss, Inc.

  11. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jinlan; Li, Xiaolu [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Feng, Yue; Zhang, Bo [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Miao, Shiying [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Linfang, E-mail: lfwangz@yahoo.com [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Na, E-mail: nawang@tsinghua.edu.cn [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

  12. Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study.

    Directory of Open Access Journals (Sweden)

    Saurabh Gupta

    Full Text Available Heat shock protein 70 (HSP70 is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70 has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state and with HSP70 protein of E. coli 70kDa DnaK (close state and relaxed them for 100 nanoseconds (ns using all-atom Molecular Dynamics (MD Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA and Minimum Distance Matrix (MDM. The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.

  13. Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans.

    Science.gov (United States)

    Rani, Aruna; Dhillon, Arun; Sharma, Kedar; Goyal, Arun

    2018-04-01

    The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α) 6 incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×10 5 ) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Interactions of Chromatin Context, Binding Site Sequence Content, and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

    OpenAIRE

    Su, Dan; Wang, Xuting; Campbell, Michelle R.; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Bell, Douglas A.

    2015-01-01

    Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, ...

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

    Science.gov (United States)

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

    1998-01-01

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

  16. PreBIND and Textomy – mining the biomedical literature for protein-protein interactions using a support vector machine

    Directory of Open Access Journals (Sweden)

    Baskin Berivan

    2003-03-01

    Full Text Available Abstract Background The majority of experimentally verified molecular interaction and biological pathway data are present in the unstructured text of biomedical journal articles where they are inaccessible to computational methods. The Biomolecular interaction network database (BIND seeks to capture these data in a machine-readable format. We hypothesized that the formidable task-size of backfilling the database could be reduced by using Support Vector Machine technology to first locate interaction information in the literature. We present an information extraction system that was designed to locate protein-protein interaction data in the literature and present these data to curators and the public for review and entry into BIND. Results Cross-validation estimated the support vector machine's test-set precision, accuracy and recall for classifying abstracts describing interaction information was 92%, 90% and 92% respectively. We estimated that the system would be able to recall up to 60% of all non-high throughput interactions present in another yeast-protein interaction database. Finally, this system was applied to a real-world curation problem and its use was found to reduce the task duration by 70% thus saving 176 days. Conclusions Machine learning methods are useful as tools to direct interaction and pathway database back-filling; however, this potential can only be realized if these techniques are coupled with human review and entry into a factual database such as BIND. The PreBIND system described here is available to the public at http://bind.ca. Current capabilities allow searching for human, mouse and yeast protein-interaction information.

  17. Substantial improvements of long-term stability in encapsulation-free WS2 using highly interacting graphene substrate

    Science.gov (United States)

    Kim, Se-Yang; Kwak, Jinsung; Kim, Jung Hwa; Lee, Jae-Ung; Jo, Yongsu; Youb Kim, Sung; Cheong, Hyeonsik; Lee, Zonghoon; Kwon, Soon-Yong

    2017-03-01

    We report the novel role of graphene substrates in obstructing the aging propagation in both the basal planes and edges of two-dimensitional sheets of transition metal dichalcogenides (TMDs). Even after 300 d in ambient air conditions, the epitaxially grown WS2/graphene samples have a clean, uniform surface without any encapsulation. We show that high crystallinity is an effective factor that determines the excellent air stability of WS2/graphene, and we present impressive experimental evidence of the relation between defects and the aging phenomena. Moreover, we reveal the strong interlayer charge interaction as an additional factor for the enhanced air stability as a result of charge transfer-induced doping. This work not only proposes a simple method to create highly stable TMDs by the selection of a suitable substrate but also paves the way for the realization of practical TMDs-based applications.

  18. Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: Advances toward treatment of hepatitis B virus infections

    Science.gov (United States)

    Shahabadi, Nahid; Falsafi, Monireh

    The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33 ± 0.2 × 104 L mol-1and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH = 34.4 kJ mol-1; ΔS = 184.32 J mol-1 K-1). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol-1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

  19. Probing into the binding interaction between medroxyprogesterone acetate and bovine serum albumin (BSA): spectroscopic and molecular docking methods.

    Science.gov (United States)

    Fang, Fang; Pan, Dong-Qi; Qiu, Min-Jie; Liu, Ting-Ting; Jiang, Min; Wang, Qi; Shi, Jie-Hua

    2016-09-01

    To further understand the mechanism of action and pharmacokinetics of medroxyprogesterone acetate (MPA), the binding interaction of MPA with bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) was studied using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, circular dichroism and molecular docking methods. The experimental results reveal that the fluorescence of BSA quenches due to the formation of MPA-BSA complex. The number of binding sites (n) and the binding constant for MPA-BSA complex are ~1 and 4.6 × 10(3)  M(-1) at 310 K, respectively. However, it can be concluded that the binding process of MPA with BSA is spontaneous and the main interaction forces between MPA and BSA are van der Waals force and hydrogen bonding interaction due to the negative values of ΔG(0) , ΔH(0) and ΔS(0) in the binding process of MPA with BSA. MPA prefers binding on the hydrophobic cavity in subdomain IIIA (site II'') of BSA resulting in a slight change in the conformation of BSA, but BSA retaining the α-helix structure. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  20. High-throughput evaluation of interactions between biomaterials, proteins and cells using patterned superhydrophobic substrates

    OpenAIRE

    Neto, Ana I.; Custódio, Catarina A.; Wenlong Song; Mano, J. F.

    2011-01-01

    We propose a new low cost platform for high-throughput analysis that permits screening the biological performance of independent combinations of biomaterials, cells and culture media. Patterned superhydrophobic flat substrates with controlled wettable spots are used to produce microarray chips for accelerated multiplexing evaluation. This work was partially supported by Fundação para a Ciência e Tecnologia (FCT) under project PTDC/FIS/68517/2006.

  1. Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

    Science.gov (United States)

    Bozeman, Trevor C; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J; Zaleski, Paul A; Wilson, W David; Hecht, Sidney M

    2012-10-31

    Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

  2. Binding interactions of convulsant and anticonvulsant gamma-butyrolactones and gamma-thiobutyrolactones with the picrotoxin receptor

    International Nuclear Information System (INIS)

    Holland, K.D.; McKeon, A.C.; Covey, D.F.; Ferrendelli, J.A.

    1990-01-01

    Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones (TBLs) are neuroactive chemicals. beta-Substituted compounds are convulsant, whereas alpha-alkyl substituted GBLs and TBLs are anticonvulsant. The structural similarities between beta-alkyl GBLs and the convulsant picrotoxinin suggested that alkyl substituted GBLs and TBLs act at the picrotoxin receptor. To test this hypothesis we examined the interactions of convulsant and anticonvulsant GBLs and TBLs with the picrotoxin, benzodiazepine and gamma-aminobutyric acid (GABA) binding sites of the GABA receptor complex. All of these convulsants and anticonvulsants studied competitively displaced 35S-t-butylbicyclophosphorothionate (35S-TBPS), a ligand that binds to the picrotoxin receptor. This inhibition of 35S-TBPS binding was not blocked by the GABA antagonist bicuculline methobromide. The convulsant GBLs and TBLs also partially inhibited [3H]muscimol binding to the GABA site and [3H]flunitrazepam binding to the benzodiazepine site, but they did so at concentrations substantially greater than those that inhibited 35S-TBPS binding. The anticonvulsant GBLs and TBLs had no effect on either [3H]muscimol or [3H]flunitrazepam binding. In contrast to the GBLs and TBLs, pentobarbital inhibited TBPS binding in a manner that was blocked by bicuculline methobromide, and it enhanced both [3H]flunitrazepam and [3H]muscimol binding. Both ethosuximide and tetramethylsuccinimide, neuroactive compounds structurally similar to GBLs, competitively displaced 35S-TBPS from the picrotoxin receptor and both compounds were weak inhibitors of [3H] muscimol binding. In addition, ethosuximide also partially diminished [3H]flunitrazepam binding. These data demonstrate that the site of action of alkyl-substituted GBLs and TBLs is different from that of GABA, barbiturates and benzodiazepines

  3. A multispectroscopic and molecular docking investigation of the binding interaction between serum albumins and acid orange dye

    Science.gov (United States)

    Naveenraj, Selvaraj; Solomon, Rajadurai Vijay; Mangalaraja, Ramalinga Viswanathan; Venuvanalingam, Ponnambalam; Asiri, Abdullah M.; Anandan, Sambandam

    2018-03-01

    The interaction of Acid Orange 10 (AO10) with bovine serum albumin (BSA) was investigated comparatively with that of human serum albumin (HSA) using multispectroscopic techniques for understanding their toxic mechanism. Further, density functional theory calculations and docking studies have been carried out to gain more insights into the nature of interactions existing between AO10 and serum albumins. The fluorescence results suggest that AO10 quenched the fluorescence of BSA through the combination of static and dynamic quenching mechanism. The same trend was followed in the interaction of AO10 with HSA. In addition to the type of quenching mechanism, the fluorescence spectroscopic results suggest that the binding occurs near the tryptophan moiety of serum albumins and the binding. AO10 has more binding affinity towards BSA than HSA. An AO10-Trp model has been created to explicitly understand the Csbnd Htbnd π interactions from Bader's quantum theory of atoms in molecules analysis which confirmed that AO10 bind more strongly with BSA than that of HSA due to the formation of three hydrogen bonds with BSA whereas it forms two hydrogen bonds in the case of HSA. These obtained results provide an in-depth understanding of the interaction of the acid azo dye AO10 with serum albumins. This interaction study provides insights into the underlying reasons for toxicity of AO10 relevant to understand its effect on bovids and humans during the blood transportation process.

  4. Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

    Science.gov (United States)

    Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

    2018-01-05

    The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

  5. The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.

    Directory of Open Access Journals (Sweden)

    Matthias Stein

    Full Text Available Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

  6. Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin.

    Science.gov (United States)

    Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi; Shi, Jie-Hua

    2017-06-01

    The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV-vis absorption spectroscopy (UV-vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady-state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10 10  L mol -1  sec -1 . This result indicates that the ENPL-BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG 0 ), enthalpic change (ΔH 0 ) and entropic change (ΔS 0 ). The binding of ENPL with BSA is an enthalpy-driven process due to |ΔH°| > |TΔS°| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub-domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α-helical secondary structure. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition.

    Science.gov (United States)

    Hara, Masatoshi; Lourido, Sebastian; Petrova, Boryana; Lou, Hua Jane; Von Stetina, Jessica R; Kashevsky, Helena; Turk, Benjamin E; Orr-Weaver, Terry L

    2018-02-26

    The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. In a previous paper (Hara et al., 2017), we demonstrated PNG activity is under tight developmental control and restricted to this transition. Here, examination of PNG specificity showed it to be a Thr-kinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and we also show that PUM is a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors. © 2018, Hara et al.

  8. Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

    Energy Technology Data Exchange (ETDEWEB)

    Eisenmesser, Elan Z.; Capodagli, Glenn; Armstrong, Geoffrey S.; Holliday, Michael; Isern, Nancy G.; Zhang, Fengli; Pegan, Scott D.

    2015-05-01

    Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove multiple proteins involved in cellular signaling such as ubiquitin (Ub) and interferon stimulated gene produce 15 (ISG15). Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the dynamic plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHV vOTU, both alone and in complex with Ub, thereby discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.

  9. Spectrofluorometric and thermal gravimetric study on binding interaction of thiabendazole with hemoglobin on epoxy-functionalized magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com; Ozmen, Mustafa

    2015-09-01

    The interaction of thiabendazole (Tbz) with hemoglobin (Hb) on epoxy-functionalized iron oxide nanoparticles was presented in this study. The binding capacity of Tbz was determined by measuring at an excitation wavelength of 299 nm using fluorescence spectroscopy. The thermodynamic parameters of the Hb–Tbz interaction were calculated from Stern–Volmer and van't Hoff equations. The values of enthalpy change, ∆H, and entropy change, ∆S, were found to be 0.20 kJ mol{sup −1} and 0.70 J mol{sup −1} K{sup −1}, respectively, which indicates that the hydrophilic interaction plays a main role in the binding process. The interaction ability was confirmed by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Also, the thermal behavior of the Hb–Tbz interaction on functionalized iron oxide nanoparticles was studied by using the thermogravimetric analysis (TGA) technique in the temperature range of 25–950 °C, and then the kinetic parameters for the thermal decomposition were determined using the Horowitz–Metzger method. - Highlights: • Hb was immobilized by covalent attachment on GPTS–SPIONs. • Interaction of Tbz with Hb–GPTS–SPIONs was studied. • Thermodynamic parameters for interaction were calculated. • Hydrophilic interaction plays a main role in the binding process.

  10. Interaction between nucleotide binding sites on chloroplast coupling factor 1 during ATP hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Leckband, D.; Hammes, G.G.

    1987-04-21

    The initial hydrolysis of radioactively-labelled CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rate constant for the first-order process is independent of substrate concentration but increased hyperbolically to a limiting value of 0.43 s/sup -1/ with increasing concentrations of free Ca/sup 2 +/. A mechanism involving a Ca/sup 2 +/-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approx. 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s/sup -1/ as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changes. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.

  11. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  12. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  13. Microbial interactions chapter: binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Genetic transformation of bacteria by DNA released from cells of a related strain is discussed. The mechanism by which the giant information-bearing molecules of DNA are transported into the bacterial cell was investigated. It was concluded that the overall process of DNA uptake consists of two main steps, binding of donor DNA to the outside of the cell and entry of the bound DNA into the cell. Each step is discussed in detail. Inasmuch as these phenomena occur at the cell surface, they are related to structures and functions of the cell wall and membrane. In addition, the development of competence, that is the formation of cell surface structures allowing DNA uptake, is examined from both a physiological and evolutionary point of view. Genetic transfer mediated by free DNA is an obvious and important form of cellular interaction. The development of competence involves another, quite distinct system of interaction between bacterial cells. Streptococcus pneumoniae, Bacillus subtilis, and Hemophilus influenzae were used as the test organisms. 259 references.

  14. Binding Cellulose and Chitosan via Intermolecular Inclusion Interaction: Synthesis and Characterisation of Gel

    Directory of Open Access Journals (Sweden)

    Jiufang Duan

    2015-01-01

    Full Text Available A novel cellulose-chitosan gel was successfully prepared in three steps: (1 ferrocene- (Fc- cellulose with degrees of substitution (DS of 0.5 wt% was synthesised by ferrocenecarboxylic acid and cellulose within dimethylacetamide/lithium chloride (DMAc/LiCl; (2 the β-cyclodextrin (β-CD groups were introduced onto the chitosan chains by reacting chitosan with epichlorohydrin in dimethyl sulphoxide and a DS of 0.35 wt%; (3 thus, the cellulose-chitosan gel was obtained via an intermolecular inclusion interaction of Fc-cellulose and β-CD-chitosan in DMA/LiCl, that is, by an intermolecular inclusion interaction, between the Fc groups of cellulose and the β-CD groups on the chitosan backbone at room temperature. The successful synthesis of Fc-cellulose and β-CD-chitosan was characterised by 13C-NMR spectroscopy. The gel based on β-CD-chitosan and Fc-cellulose was formed under mild conditions which can engender autonomous healing between cut surfaces after 24 hours: the gel cannot self-heal while the cut surfaces were coated with a solution of a competitive guest (adamantane acid. The cellulose-chitosan complex made by this method underwent self-healing. Therefore, this study provided a novel method of expanding the application of chitosan by binding it with another polymer.

  15. Unraveling Prion Protein Interactions with Aptamers and Other PrP-Binding Nucleic Acids.

    Science.gov (United States)

    Macedo, Bruno; Cordeiro, Yraima

    2017-05-17

    Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders that affect humans and other mammals. The etiologic agents common to these diseases are misfolded conformations of the prion protein (PrP). The molecular mechanisms that trigger the structural conversion of the normal cellular PrP (PrP C ) into the pathogenic conformer (PrP Sc ) are still poorly understood. It is proposed that a molecular cofactor would act as a catalyst, lowering the activation energy of the conversion process, therefore favoring the transition of PrP C to PrP Sc . Several in vitro studies have described physical interactions between PrP and different classes of molecules, which might play a role in either PrP physiology or pathology. Among these molecules, nucleic acids (NAs) are highlighted as potential PrP molecular partners. In this context, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology has proven extremely valuable to investigate PrP-NA interactions, due to its ability to select small nucleic acids, also termed aptamers, that bind PrP with high affinity and specificity. Aptamers are single-stranded DNA or RNA oligonucleotides that can be folded into a wide range of structures (from harpins to G-quadruplexes). They are selected from a nucleic acid pool containing a large number (10 14 -10 16 ) of random sequences of the same size (~20-100 bases). Aptamers stand out because of their potential ability to bind with different affinities to distinct conformations of the same protein target. Therefore, the identification of high-affinity and selective PrP ligands may aid the development of new therapies and diagnostic tools for TSEs. This review will focus on the selection of aptamers targeted against either full-length or truncated forms of PrP, discussing the implications that result from interactions of PrP with NAs, and their potential advances in the studies of prions. We will also provide a critical evaluation

  16. Explaining an Unusually Fast Parasitic Enzyme: Folate Tail-Binding Residues Dictate Substrate Positioning and Catalysis in Cryptosporidium hominis Thymidylate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Martucci,W.; Vargo, M.; Anderson, K.

    2008-01-01

    The essential enzyme TS-DHFR from Cryptosporidium hominis undergoes an unusually rapid rate of catalysis at the conserved TS domain, facilitated by two nonconserved residues, Ala287 and Ser290, in the folate tail-binding region. Mutation of these two residues to their conserved counterparts drastically affects multiple steps of the TS catalytic cycle. We have determined the crystal structures of all three mutants (A287F, S290G, and A287F/S290G) in complex with active site ligands dUMP and CB3717. The structural data show two effects of the mutations: an increased distance between the ligands in the active site and increased flexibility of the folate ligand in the partially open enzyme state that precedes conformational change to the active catalytic state. The latter effect is able to be rescued by the mutants containing the A287F mutation. In addition, the conserved water network of TS is altered in each of the mutants. The structural results point to a role of the folate tail-binding residues in closely positioning ChTS ligands and restricting ligand flexibility in the partially open state to allow for a rapid transition to the active closed state and enhanced rate of catalysis. These results provide an explanation on how folate tail-binding residues at one end of the active site affect long-range interactions throughout the TS active site and validate these residues as targets for species-specific drug design.

  17. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  18. Spectroscopic study of interaction between osthole and human serum albumin: Identification of possible binding site of the compound

    Energy Technology Data Exchange (ETDEWEB)

    Bijari, Nooshin [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Shokoohinia, Yalda [Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza; Ranjbar, Samira; Parvaneh, Shahram [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Moieni-Arya, Maryam [Student Research Committee, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

    2013-11-15

    The studies on the interaction between human serum albumin (HSA) and drugs have been an interesting research field in life science, chemistry and clinical medicine. Osthole possesses a variety of pharmacological activities including anti-tumor, anti-inflammation, anti-seizure, anti-hyperlipidemic and anti-osteoporosis effects. The interaction of osthole with HSA and its binding site in HSA by spectroscopic methods is the subject of this work. By monitoring the intrinsic fluorescence of the single Trp{sub 214} residue and performing site markers displacement measurements, the specific binding of osthole in the vicinity of Sudlow's site I of HSA has been clarified. The changes in the secondary structure of HSA after its complexation with ligand were studied with CD spectroscopy, which indicate that osthole induced only a slight decrease in the helix structural content of the protein. In addition, the mean distance between osthole and HSA fluorophores is estimated to be 4.96 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Osthole can quench the intrinsic fluorescence of HSA by dynamic quenching, and analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play an important role in the stabilizing of the complex. Increase of protein surface hydrophobicity (PSH) was also observed upon the osthole binding. -- Highlights: • Hydrophobic interactions play an important role in osthole–HSA interaction. • Sudlow's I site is possible binding site of osthole. • Osthole inhibits esterase activity of HSA. • Osthole binding induces no gross protein structural changes.

  19. Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

    International Nuclear Information System (INIS)

    Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini

    2011-01-01

    Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres

  20. Auger electron spectroscopy study on interaction between aluminum thin layers and uranium substrate

    International Nuclear Information System (INIS)

    Zhou Wei; Liu Kezhao; Yang Jiangrong; Xiao Hong; Jiang Chunli; Lu Lei

    2005-01-01

    Aluminum thin layers on uranium were prepared by sputter deposition at room temperature in ultra high vacuum analysis chamber. Interaction between U and Al, and growth mode were investigated by Auger electron spectroscopy (AES) and electron energy loss spectroscopy (EELS). It is shown that Al thin film growth follows the volmer-weber (VW) mode. At room temperature, Al and U interact with each other, resulting in interdiffusion action and formation of U-Al alloys at U/Al interface. Annealing promotes interaction and interdiffusion between U and Al, and UAl x maybe formed at interface. (authors)

  1. Interaction of fisetin with human serum albumin by fluorescence, circular dichroism spectroscopy and DFT calculations: binding parameters and conformational changes

    International Nuclear Information System (INIS)

    Matei, Iulia; Ionescu, Sorana; Hillebrand, Mihaela

    2011-01-01

    The interaction between fisetin, an antioxidant and neuroprotective flavonoid, and human serum albumin (HSA) is investigated by means of fluorescence (steady-state, synchronous, time-resolved) and circular dichroism (CD) spectroscopy. The formation of a 1:1 complex with a constant of about 10 5 M -1 was evidenced. Foerster's resonance energy transfer and competitive binding with site markers warfarin and ibuprofen were considered and discussed. Changes in the CD band of HSA indicate a decrease in the α-helix content upon binding. An induced CD signal for bound fisetin was observed and rationalized in terms of density functional theory calculations. - Highlights: → Fisetin-BSA system was studied by fluorescence spectroscopy. → Binding parameters, association constant and number of sites were estimated. → Binding site of fisetin was identified by competitive experiments. → Conformational changes in HSA and fisetin were evidenced by circular dichroism. → TDDFT calculated CD spectra supported the experimental data.

  2. Interaction of fisetin with human serum albumin by fluorescence, circular dichroism spectroscopy and DFT calculations: binding parameters and conformational changes

    Energy Technology Data Exchange (ETDEWEB)

    Matei, Iulia; Ionescu, Sorana [Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina Elisabeta 4-12, 030018 Bucharest (Romania); Hillebrand, Mihaela, E-mail: mihh@gw-chimie.math.unibuc.ro [Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina Elisabeta 4-12, 030018 Bucharest (Romania)

    2011-08-15

    The interaction between fisetin, an antioxidant and neuroprotective flavonoid, and human serum albumin (HSA) is investigated by means of fluorescence (steady-state, synchronous, time-resolved) and circular dichroism (CD) spectroscopy. The formation of a 1:1 complex with a constant of about 10{sup 5} M{sup -1} was evidenced. Foerster's resonance energy transfer and competitive binding with site markers warfarin and ibuprofen were considered and discussed. Changes in the CD band of HSA indicate a decrease in the {alpha}-helix content upon binding. An induced CD signal for bound fisetin was observed and rationalized in terms of density functional theory calculations. - Highlights: > Fisetin-BSA system was studied by fluorescence spectroscopy. > Binding parameters, association constant and number of sites were estimated. > Binding site of fisetin was identified by competitive experiments. > Conformational changes in HSA and fisetin were evidenced by circular dichroism. > TDDFT calculated CD spectra supported the experimental data.

  3. Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism.

    Science.gov (United States)

    Nocek, Boguslaw; Reidl, Cory; Starus, Anna; Heath, Tahirah; Bienvenue, David; Osipiuk, Jerzy; Jedrzejczak, Robert; Joachimiak, Andrzej; Becker, Daniel P; Holz, Richard C

    2018-02-06

    The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn-Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes.

  4. A cation-π interaction at a phenylalanine residue in the glycine receptor binding site is conserved for different agonists

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Hanek, Ariele P; Price, Kerry L

    2011-01-01

    . In the current study, we investigated whether the lower efficacy agonists of the human GlyR β-alanine and taurine also form cation-π interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-π interactions between Phe159 and the amino groups of β-alanine and taurine....... The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that β-alanine and taurine are orientated subtly differently in the binding pocket, with their amino groups further from Phe159 than that of glycine. These data therefore show that similar agonists can have...... similar but not identical orientations and interactions in the binding pocket and provide a possible explanation for the lower potencies of β-alanine and taurine....

  5. Increased interaction with insulin receptor substrate 1, a novel abnormality in insulin resistance and type 2 diabetes

    DEFF Research Database (Denmark)

    Caruso, Michael; Ma, Danjun; Msallaty, Zaher

    2014-01-01

    Insulin receptor substrate 1 (IRS1) is a key mediator of insulin signal transduction. Perturbations involving IRS1 complexes may lead to the development of insulin resistance and type 2 diabetes (T2D). Surprisingly little is known about the proteins that interact with IRS1 in humans under health...... in obesity and T2D in humans, provides new insights into the molecular mechanism of insulin resistance and identifies new targets for T2D drug development....... and disease conditions. We used a proteomic approach to assess IRS1 interaction partners in skeletal muscle from lean healthy control subjects (LCs), obese insulin-resistant nondiabetic control subjects (OCs), and participants with T2D before and after insulin infusion. We identified 113 novel endogenous IRS1...

  6. Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver.

    Science.gov (United States)

    Cannon, J R; Eacho, P I

    1991-01-01

    Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation. Images Fig. 2. PMID:1747111

  7. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Science.gov (United States)

    Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  8. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Directory of Open Access Journals (Sweden)

    Ting Lu

    Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  9. Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

    Science.gov (United States)

    Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

    2014-01-01

    The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

  10. Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

    Science.gov (United States)

    Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

    1998-01-01

    Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

  11. The interaction of quinones, herbicides and bicarbonate with their binding environment at the acceptor side of photosystem II in photosynthesis

    NARCIS (Netherlands)

    Vermaas, W.F.J.

    1984-01-01

    In this thesis experiments are described which are directed towards a further characterization of the interaction of the native bound plastoquinone Q B , artificial quinones, herbicides and bicarbonate with their binding environment at the acceptor side of Photosystem II in

  12. Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

    Science.gov (United States)

    Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

    2016-03-01

    The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

  13. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module

    DEFF Research Database (Denmark)

    Salcini, A E; Confalonieri, S; Doria, M

    1997-01-01

    EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct...

  14. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

    Science.gov (United States)

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-01-01

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

  15. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

    Science.gov (United States)

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-11-27

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Stereochemistry of charged nitrogen-aromatic interactions and its involvement in ligand-receptor binding

    Science.gov (United States)

    Verdonk, Marcel L.; Boks, Gertjan J.; Kooijman, Huub; Kanters, Jan A.; Kroon, Jan

    1993-04-01

    Recently, new evidence was found for the involvement of charged nitrogen-aromatic interactions in ligand-receptor binding. In this study we report two favourable orientations of a phenyl ring with respect to a R-N+(CH3)3 group, based on crystal structure statistics from the Cambridge Structural Database. In the first orientation, the phenyl ring is situated in between the substituents at about 4.5 Å from the nitrogen atom, and the ring is approximately oriented on the sphere around the nitrogen atom. In the second orientation, the phenyl ring is situated in the same direction as one of the N-C bonds at about 6.0 Å from the nitrogen atom, and the ring is tilted with respect to the sphere around the nitrogen atom. The same two orientations were also found in the crystal structures of three ligand-receptor complexes, which implies that these orientations probably play a major role in molecular recognition mechanisms.

  17. The role of the lysyl binding site of tissue-type plasminogen activator in the interaction with a forming fibrin clot

    NARCIS (Netherlands)

    Bakker, A.H.F.; Weening-Verhoeff, E.J.D.; Verheijen, J.H.

    1995-01-01

    To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the

  18. Binding interaction of ramipril with bovine serum albumin (BSA): Insights from multi-spectroscopy and molecular docking methods.

    Science.gov (United States)

    Shi, Jie-Hua; Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi

    2016-11-01

    The binding interaction between a typical angiotensin-converting enzyme inhibitor (ACEI), ramipril, and a transport protein, bovine serum albumin (BSA), was studied in vitro using UV-vis absorption spectroscopy, steady-state fluorescence spectroscopic titration, synchronous fluorescence spectroscopy, three dimensional fluorescence spectroscopy, circular dichroism and molecular docking under the imitated physiological conditions (pH=7.4). The experimental results suggested that the intrinsic fluorescence of BSA was quenched by ramipril thought a static quenching mechanism, indicating that the stable ramipril-BSA complex was formed by the intermolecular interaction. The number of binding sites (n) and binding constant of ramipril-BSA complex were about 1 and 3.50×10 4 M -1 at 298K, respectively, suggesting that there was stronger binding interaction of ramipril with BSA. The thermodynamic parameters together with molecular docking study revealed that both van der Waal's forces and hydrogen bonding interaction dominated the formation of the ramipril-BSA complex and the binding interaction of BSA with ramipril is enthalpy-driven processes due to |ΔH°|>|TΔS°| and ΔG°<0. The spatial distance between ramipril and BSA was calculated to be 3.56nm based on Förster's non-radiative energy transfer theory. The results of the competitive displacement experiments and molecular docking confirmed that ramipril inserted into the subdomain IIA (site I) of BSA, resulting in a slight change in the conformation of BSA but BSA still retained its secondary structure α-helicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Interaction of aconitine with bovine serum albumin and effect of atropine sulphate and glycyrrhizic acid on the binding

    International Nuclear Information System (INIS)

    Huang Yun; Cui Lijian; Wang Jianming; Huo Kun; Chen Chen; Zhan Wenhong; Wang Yongli

    2012-01-01

    The interaction of aconitine with bovine serum albumin (BSA) and effect of atropine sulphate and glycyrrhizic acid on binding constant, binding sites, and conformation were studied in an aqueous buffer solution (pH 7.40) by ultraviolet absorption and fluorescence spectroscopy. The study results show that aconitine quenched the endogenous fluorescence of BSA via a dynamic quenching procedure. Predominant intermolecular forces between aconitine and BSA were hydrophobic interactions, which stabilized the complex of aconitine–BSA. The distance between the donor and acceptor was 2.62 nm. The conformation of BSA was investigated by synchronous fluorescence techniques, indicating that the microenvironment around tryptophan (Trp) residues was changed. Furthermore, with the addition of atropine sulphate or glycyrrhizic acid, binding constant and the number of binding sites of aconitine to BSA were decreased, and the conformation had no change, which provide an important theoretical support for aconitine detoxification by atropine sulphate and glycyrrhizic acid. - Highlights: ► Effect of atropine or glycyrrhizic acid on aconitine–BSA binding. ► UV–vis absorption and fluorescence spectroscopic techniques used. ► Aconitine quenched BSA fluorescence via dynamic quenching with r=2.62 nm. ► Atropine sulphate and glycyrrhizic acid decreased K A and n of aconitine–BSA. ► Support for aconitine detoxification by atropine and glycyrrhizic acid.

  20. Effects of varying oxygen partial pressure on molten silicon-ceramic substrate interactions

    Science.gov (United States)

    Ownby, D. P.; Barsoum, M. W.

    1980-01-01

    The silicon sessile drop contact angle was measured on hot pressed silicon nitride, silicon nitride coated on hot pressed silicon nitride, silicon carbon coated on graphite, and on Sialon to determine the degree to which silicon wets these substances. The post-sessile drop experiment samples were sectioned and photomicrographs were taken of the silicon-substrate interface to observe the degree of surface dissolution and degradation. Of these materials, silicon did not form a true sessile drop on the SiC on graphite due to infiltration of the silicon through the SiC coating, nor on the Sialon due to the formation of a more-or-less rigid coating on the liquid silicon. The most wetting was obtained on the coated Si3N4 with a value of 42 deg. The oxygen concentrations in a silicon ribbon furnace and in a sessile drop furnace were measured using the protable thoria-yttria solid solution electrolyte oxygen sensor. Oxygen partial pressures of 10 to the minus 7 power atm and 10 to the minus 8 power atm were obtained at the two facilities. These measurements are believed to represent nonequilibrium conditions.

  1. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

    Directory of Open Access Journals (Sweden)

    Yumiko Urano-Tashiro

    Full Text Available Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2 of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N or Arg365 to Asn (R365N substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins.

  2. Asymmetric binding of histone H1 stabilizes MMTV nucleosomes and the interaction of progesterone receptor with the exposed HRE.

    Science.gov (United States)

    Vicent, Guillermo P; Meliá, María J; Beato, Miguel

    2002-11-29

    Packaging of mouse mammary tumor virus (MMTV) promoter sequences in nucleosomes modulates access of DNA binding proteins and influences the interaction among DNA bound transcription factors. Here we analyze the binding of histone H1 to MMTV mononucleosomes assembled with recombinant histones and study its influence on nucleosome structure and stability as well as on progesterone receptor (PR) binding to the hormone responsive elements (HREs). The MMTV nucleosomes can be separated into three main populations, two of which exhibited precise translational positioning. Histone H1 bound preferentially to the 5' distal nucleosomal DNA protecting additional 27-28 nt from digestion by micrococcal nuclease. Binding of histone H1 was unaffected by prior crosslinking of protein and DNA in nucleosomes with formaldehyde. Neither the translational nor the rotational nucleosome positioning was altered by histone H1 binding, but the nucleosomes were stabilized as judged by the kinetics of nuclease cleavage. Unexpectedly, binding of recombinant PR to the exposed distal HRE-I in nucleosomes was enhanced in the presence of histone H1, as demonstrated by band shift and footprinting experiments. This enhanced PR affinity may contribute to the reported positive effect of histone H1 on the hormonal activation of MMTV reporter genes.

  3. Mechanism of hydrogen peroxide dismutation by a dimanganese catalase mimic: dominant role of an intramolecular base on substrate binding affinity and rate acceleration.

    Science.gov (United States)

    Boelrijk, A E; Dismukes, G C

    2000-07-10

    Several modifications of the manganese coordination environment and oxidation states of a family of synthetic dimanganese complexes have been introduced in search of the structural features that promote high rates of hydrogen peroxide dismutation (catalase activity). The X-ray structure of reduced catalase (T thermophilus) reveals a dimanganese(II,II) site linked by three bridges: mu 13-glutamate-, mu-OH-, and mu-OH2. The roles of a bridging hydroxide vs mu-aqua and the carboxylate have been examined in the reduced Mn2(II,II) complexes, [(L1,2)Mn2(mu-O2CCH3)(mu-X)]2+ for X- = OH- (7A) or X = H2O (1-4), and their oxidized Mn2(III,III) analogues, [(L1,2)Mn2(mu-O)(O2CCH3)(OH)]+ (6) (L1 is N,N,N',N'-tetrakis(2-methylenebenzamidazolyl)-1,3-diaminopropan- 2-ol, and L2 is the tetrakis-N-ethylated analogue of L1, which has all amine protons replaced by ethyl groups). The steady-state catalase rate is first-order in concentration of both substrate and reduced catalyst and saturates at high peroxide concentrations in all cases, confirming peroxide/catalyst complex formation. No catalyst decomposition is seen after > 2000 turnovers. Catalysis proceeds via a ping-pong mechanism between the Mn2(II,II/III,III) redox states, involving complexes 6 and 7A/7A'. The Mn2(III,IV) oxidation state was not active in catalase activity. Replacement of the mu-aqua bridge by mu-hydroxide eliminates a kinetic lag phase in production of the O2 product, increases the affinity for substrate peroxide in the rate-limiting step as seen by a 5-fold. decrease in the Michaelis constant (KM), and accelerates the maximum rate (kcat) by 65-fold The kinetic and spectroscopic data are consistent with substrate deprotonation by the hydroxide bridge, yielding a hydroperoxyl bridge coordinated between the Mn ions (mu, eta 2 geometry, "end-on") as the basis for catalysis: mu-OH- + H2O2-->mu-O2H- + H2O. Binding of a second hydroxide ion to 7A causes a further increase in kcat by 4-fold with no further change in

  4. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  5. Interaction study on bovine serum albumin physically binding to silver nanoparticles: Evolution from discrete conjugates to protein coronas

    Science.gov (United States)

    Guo, Jun; Zhong, Ruibo; Li, Wanrong; Liu, Yushuang; Bai, Zhijun; Yin, Jun; Liu, Jingran; Gong, Pei; Zhao, Xinmin; Zhang, Feng

    2015-12-01

    The nanostructures formed by inorganic nanoparticles together with organic molecules especially biomolecules have attracted increasing attention from both industries and researching fields due to their unique hybrid properties. In this paper, we systemically studied the interactions between amphiphilic polymer coated silver nanoparticles and bovine serum albumins by employing the fluorescence quenching approach in combination with the Stern-Volmer and Hill equations. The binding affinity was determined to 1.30 × 107 M-1 and the interaction was spontaneously driven by mainly the van der Waals force and hydrogen-bond mediated interactions, and negatively cooperative from the point of view of thermodynamics. With the non-uniform coating of amphiphilic polymer, the silver nanoparticles can form protein coronas which can become discrete protein-nanoparticle conjugates when controlling their molar ratios of mixing. The protein's conformational changes upon binding nanoparticles was also studied by using the three-dimensional fluorescence spectroscopy.

  6. Neural substrates of interactive musical improvisation: an FMRI study of 'trading fours' in jazz.

    Directory of Open Access Journals (Sweden)

    Gabriel F Donnay

    Full Text Available Interactive generative musical performance provides a suitable model for communication because, like natural linguistic discourse, it involves an exchange of ideas that is unpredictable, collaborative, and emergent. Here we show that interactive improvisation between two musicians is characterized by activation of perisylvian language areas linked to processing of syntactic elements in music, including inferior frontal gyrus and posterior superior temporal gyrus, and deactivation of angular gyrus and supramarginal gyrus, brain structures directly implicated in semantic processing of language. These findings support the hypothesis that musical discourse engages language areas of the brain specialized for processing of syntax but in a manner that is not contingent upon semantic processing. Therefore, we argue that neural regions for syntactic processing are not domain-specific for language but instead may be domain-general for communication.

  7. Neural substrates of interactive musical improvisation: an FMRI study of 'trading fours' in jazz.

    Science.gov (United States)

    Donnay, Gabriel F; Rankin, Summer K; Lopez-Gonzalez, Monica; Jiradejvong, Patpong; Limb, Charles J

    2014-01-01

    Interactive generative musical performance provides a suitable model for communication because, like natural linguistic discourse, it involves an exchange of ideas that is unpredictable, collaborative, and emergent. Here we show that interactive improvisation between two musicians is characterized by activation of perisylvian language areas linked to processing of syntactic elements in music, including inferior frontal gyrus and posterior superior temporal gyrus, and deactivation of angular gyrus and supramarginal gyrus, brain structures directly implicated in semantic processing of language. These findings support the hypothesis that musical discourse engages language areas of the brain specialized for processing of syntax but in a manner that is not contingent upon semantic processing. Therefore, we argue that neural regions for syntactic processing are not domain-specific for language but instead may be domain-general for communication.

  8. Interaction of cw CO2 laser radiation with plasma near-metallic substrate surface

    Science.gov (United States)

    Azharonok, V. V.; Astapchik, S. A.; Zabelin, Alexandre M.; Golubev, Vladimir S.; Golubev, V. S.; Grezev, A. N.; Filatov, Igor V.; Chubrik, N. I.; Shimanovich, V. D.

    2000-07-01

    Optical and spectroscopic methods were used in studying near-surface plasma that is formed under the effect CW CO2 laser of (2- 5)x106W/cm2 power density upon stainless steel in He and Ar shielding gases. The variation of plume spatial structure with time has been studied, the outflow of gas-vapor jets from the interaction area has been characterized. The spectra of plasma plume pulsations have been obtained for the frequency range Δf = 0-1 MHz. The temperature and electron concentration of plasma plume have been found under radiation effect upon the target of stainless steel. Consideration has been given to the most probable mechanisms of CW laser radiation-metal non-stationary interaction.

  9. The van der Waals interaction of microparticles with a substrate characterized by a nonlocal response

    International Nuclear Information System (INIS)

    Dorofeyev, Illarion

    2007-01-01

    The van der Waals energy of the system constituted by a microparticle and a solid surface characterized by a nonlocal response is calculated taking into account an influence of another microparticle. A saturation of the dispersion interaction at short distances from the surface both for the spectral density of energy and for the total energy is shown. The known McLachlan expression for the pair and triple energies in the case of local media directly follows from the obtained general expression

  10. Study of binding interaction between anthelmintic 2, 3-dihydroquinazolin-4-ones with bovine serum albumin by spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Hemalatha, K.; Madhumitha, G., E-mail: madhumitha.g@vit.ac.in

    2016-10-15

    A new series of brominated derivatives of 2, 3-dihydroquinazolin-4(1H)-one were synthesized and their structures were confirmed using IR, NMR and mass spectra. The synthesized derivatives were screened for their in vitro anthelmintic activity. The investigations on interaction of the bioactive compound, 2i with bovine serum albumin (BSA) were evaluated. The quenching mechanism of the compound, 2i was deduced based on the results of Stern–Volmer equation. The number of binding site, prediction of binding site region and the changes in the secondary structure of protein were predicted using various spectroscopic studies.

  11. Synthetic protease substrate n-benzoyl-L-argininyl-p-nitroanilide activates specific binding of [3H]estradiol to a protein in rat pancreas: relationship of structure to activity

    International Nuclear Information System (INIS)

    Grossman, A.

    1984-01-01

    N-benzoyl-L-argininyl-p-nitroanilide (BAN), a synthetic substrate for trypsin-like proteolytic enzymes, is a potent activator of [ 3 H]estradiol-binding to a protein present in rat pancreas. When partially purified, this protein is almost devoid of [ 3 H]estradiol-binding activity in the absence of an endogenous accessory factor. BAN can mimic the natural coligand in this steroid binding reaction. The effect of BAN is specific since a number of derivatives of this substance are inactive or may even inhibit steroid binding. It is unlikely that BAN exerts this stimulatory action indirectly, possibly by preventing proteolytic inactivation of the [ 3 H]estradiol-binding protein, since preincubation of the protein in the absence of BAN resulted neither in reduced rate, nor extent, of steroid binding following BAN addition. Also, a number of protease inhibitors had no effect on the binding reaction. Of those inhibitors tested, only antipain significantly enhanced binding of [ 3 H]estradiol, but only about 20 percent as effectively as BAN. 13 references, 1 figure, 2 tables

  12. DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

    Science.gov (United States)

    Keyamura, Kenji; Katayama, Tsutomu

    2011-08-19

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

  13. DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

    Science.gov (United States)

    Keyamura, Kenji; Katayama, Tsutomu

    2011-01-01

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

  14. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet G s protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al +3 and F - agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF - 4 strongly inhibited 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  15. Precise Placement of Metallic Nanowires on a Substrate by Localized Electric Fields and Inter-Nanowire Electrostatic Interaction

    Directory of Open Access Journals (Sweden)

    U Hyeok Choi

    2017-10-01

    Full Text Available Placing nanowires at the predetermined locations on a substrate represents one of the significant hurdles to be tackled for realization of heterogeneous nanowire systems. Here, we demonstrate spatially-controlled assembly of a single nanowire at the photolithographically recessed region at the electrode gap with high integration yield (~90%. Two popular routes, such as protruding electrode tips and recessed wells, for spatially-controlled nanowire alignment, are compared to investigate long-range dielectrophoretic nanowire attraction and short-range nanowire-nanowire electrostatic interaction for determining the final alignment of attracted nanowires. Furthermore, the post-assembly process has been developed and tested to make a robust electrical contact to the assembled nanowires, which removes any misaligned ones and connects the nanowires to the underlying electrodes of circuit.

  16. Interplay of adsorbate-adsorbate and adsorbate-substrate interactions in self-assembled molecular surface nanostructures

    DEFF Research Database (Denmark)

    Schnadt, Joachim; Xu, Wei; Vang, Ronnie Thorbjørn

    2010-01-01

    a large tolerance to monatomic surface steps on the Ag(110) surface. The observed behaviour is explained in terms of strong intermolecular hydrogen bonding and a strong surface-mediated directionality, assisted by a sufficient degree of molecular backbone flexibility. In contrast, the same kind of step......-edge crossing is not observed when the molecules are adsorbed on the isotropic Ag(111) or more reactive Cu(110) surfaces. On Ag(111), similar 1-D assemblies are formed to those on Ag(110), but they are oriented along the step edges. On Cu(110), the carboxylic groups of NDCA are deprotonated and form covalent...... bonds to the surface, a situation which is also achieved on Ag(110) by annealing to 200 degrees C. These results show that the formation of particular self-assembled molecular nanostructures depends significantly on a subtle balance between the adsorbate-adsorbate and adsorbate-substrate interactions...

  17. Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein-Protein Interactions.

    Science.gov (United States)

    Kang, Beom Sik; Pugalendhi, GaneshKumar; Kim, Ku-Jin

    2017-10-13

    Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

  18. Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein–Protein Interactions

    Directory of Open Access Journals (Sweden)

    Beom Sik Kang

    2017-10-01

    Full Text Available Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

  19. Interaction of 3D dewetting nanodroplets on homogeneous and chemically heterogeneous substrates

    International Nuclear Information System (INIS)

    Asgari, M; Moosavi, A

    2014-01-01

    Long-time interaction of dewetting nanodroplets is investigated using a long-wave approximation method. Although three-dimensional (3D) droplets evolution dynamics exhibits qualitative behavior analogous to two-dimensional (2D) dynamics, there is an extensive quantitative difference between them. 3D dynamics is substantially faster than 2D dynamics. This can be related to the larger curvature and, as a consequence, the larger Laplace pressure difference between the droplets in 3D systems. The influence of various chemical heterogeneities on the behavior of droplets has also been studied. In the case of gradient surfaces, it is shown how the gradient direction could change the dynamics. For a chemical step located between the droplets, the dynamics is enhanced or weakened depending on the initial configuration of the system. (paper)

  20. A microcalorimetry and binding study on interaction of dodecyl trimethylammonium bromide with wigeon hemoglobin

    International Nuclear Information System (INIS)

    Bordbar, A.K.; Moosavi-Movahedi, A.A.; Amini, M.K.

    2003-01-01

    The thermodynamic parameters for the binding of dodecyl trimethylammonium bromide (DTAB) with wigeon hemoglobin (Hb) in aqueous solution at various pH and 27 deg. C have been measured by equilibrium dialysis and titration microcalorimetry techniques. The Scatchard plots represent unusual features at neutral and alkaline pH and specific binding at acidic pH. This leads us to analyze the binding data by fitting the data to the Hill equation for multiclasses of binding sites. The best fit was obtained with the equation for one class at acidic pH and two classes at neutral and alkaline pH. The thermodynamic analysis of the binding process shows that the strength of binding at neutral pH is more than these at other pH values. This can be related to the more accessible hydrophobic surface area of wigeon hemoglobin at this pH. The endothermic enthalpy data which was measured by microcalorimetry confirms the binding data analysis and represents the more regular and stable structure of wigeon hemoglobin at neutral pH

  1. NMR studies of DNA oligomers and their interactions with minor groove binding ligands

    Energy Technology Data Exchange (ETDEWEB)

    Fagan, Patricia A. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I•C base pairs are functional analogs of A•T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1 ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.

  2. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  3. Interaction of cadmium with atrial natriuretic factor receptors: Ligand binding and cellular processing

    International Nuclear Information System (INIS)

    Giridhar, J.; Rathinavelu, A.; Isom, G.E.

    1990-01-01

    ANF is a peptide hormone secreted by the heart and produces potent diuresis and vascular smooth muscle relaxation. It is well known that Cd produces cardiovascular toxicity and is implicated in the pathogenesis of hypertension. Hence the effects of Cd on ANF receptor dynamics and ligand binding were studied in PC12 cells. Receptor internalization using 125 I-ANF as the ligand at 37 degree C displayed a decrease in endocytic rate constants (ERC) when either preincubated with Cd (500 μM for 30 min, ERC = 0.183/min) or coincubated with Cd (500 μM, ERC = 0.196) when compared to control value (ERC = 0.259/min). Ligand binding ( 125 I-ANF) was changed by Cd as reflected by a decrease in the number of binding sites/cell in both Cd preincubated (Kd = 3.81 x 10 -10 M, B max = 1 x 10 -10 M, binding sites/cell = 9333) and coincubated cells (Kd = 1.76 x 10 -10 M, B max = 3.92 x 10 -11 M, binding sites/cell = 5960) from control (Kd = 3.87 x 10 -10 M, B max = 9.58 x 10 -11 M, binding sites/cell = 12141). Photoaffinity labelling with 125 I-ANF as the ligand was used to measure receptor subtype binding. Coincubation of cells with Cd (500 μM) and ligand decreased both high and low mol. wt. receptor binding, whereas preincubation with Cd (500μM) for 60 min produced a slight decrease in binding of both receptor subtypes. These results indicate that the cardiovascular toxicity of Cd may be partially mediated by altered ANF receptor function

  4. Investigation of high temperature reactions on solid substrates with Rutherford backscattering spectrometry: interaction of palladium with selenium on heated graphite surfaces

    International Nuclear Information System (INIS)

    Majidi, V.; Robertson, J.D.

    1991-01-01

    Selenium and palladium interactions on heated pyrolytically coated graphite substrates were investigated using Rutherford backscattering spectrometry. The studies were performed using selenium alone, palladium alone, and a combination of selenium and palladium deposited on the graphite substrates. The results indicate that palladium instantaneously stabilizes selenium at ambient temperatures and prevents the diffusion of selenium into the graphite. As the substrate is heated, temperature dependent diffusion of all analytes into the graphite is observed. Furthermore, it appears that the stabilization of selenium is due to the formation of a stoichiometric compound with palladium and oxygen. This compound decomposes at a temperature between 1070 and 1770 K. (author)

  5. Investigation of high temperature reactions on solid substrates with Rutherford backscattering spectrometry: interaction of palladium with selenium on heated graphite surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Majidi, V.; Robertson, J.D. (Kentucky Univ., Lexington, KY (United States). Dept. of Chemistry)

    1991-01-01

    Selenium and palladium interactions on heated pyrolytically coated graphite substrates were investigated using Rutherford backscattering spectrometry. The studies were performed using selenium alone, palladium alone, and a combination of selenium and palladium deposited on the graphite substrates. The results indicate that palladium instantaneously stabilizes selenium at ambient temperatures and prevents the diffusion of selenium into the graphite. As the substrate is heated, temperature dependent diffusion of all analytes into the graphite is observed. Furthermore, it appears that the stabilization of selenium is due to the formation of a stoichiometric compound with palladium and oxygen. This compound decomposes at a temperature between 1070 and 1770 K. (author).

  6. Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

    Science.gov (United States)

    Chen, Yaozong; Li, Tin Lok; Lin, Xingbang; Li, Xin; Li, Xiang David; Guo, Zhihong

    2017-07-21

    o -Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

    Science.gov (United States)

    Chabrol, Eric; Nurisso, Alessandra; Daina, Antoine; Vassal-Stermann, Emilie; Thepaut, Michel; Girard, Eric; Vivès, Romain R; Fieschi, Franck

    2012-01-01

    Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs), a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+)-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions.

  8. Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

    Directory of Open Access Journals (Sweden)

    Eric Chabrol

    Full Text Available Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs, a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions.

  9. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  10. The effect of including tensor forces in nucleon-nucleon interaction on three-nucleon binding energy

    International Nuclear Information System (INIS)

    Osman, A.; Ramadan, S.

    1986-01-01

    Separable two-body interactions are used in considering the three-nucleon problem. The nucleon-nucleon potentials are taken to include attraction and repulsion as well as tensor forces. The separable approximation is used in order to investigate the effect of the tensor forces. The separable expansion is introduced in the three-nucleon problem, by which the Faddeev equations are reduced to a well-behaved set of coupled integral equations. Numerical calculations are carried out for the obtained integral equations using potential functions of the Yamaguchi, Gaussian, Takabin, Mongan and Reid forms. The present calculated values of the binding energies of the 3 H and 3 He nuclei are in good agreement with the experimental values. The effect of including the tensor forces in the nucleon-nucleon interactions is found to improve the three-nucleon binding energy by about 4.490% to 8.324%. 37 refs., 2 tabs. (author)

  11. Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

    Science.gov (United States)

    Ueda, I; Yamanaka, M

    1997-04-01

    Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency.

  12. Structural characterization of the binding interactions of various endogenous estrogen metabolites with human estrogen receptor α and β subtypes: a molecular modeling study.

    Directory of Open Access Journals (Sweden)

    Pan Wang

    Full Text Available In the present study, we used the molecular docking approach to study the binding interactions of various derivatives of 17β-estradiol (E2 with human estrogen receptor (ER α and β. First, we determined the suitability of the molecular docking method to correctly predict the binding modes and interactions of two representative agonists (E2 and diethylstilbesterol in the ligand binding domain (LBD of human ERα. We showed that the docked structures of E2 and diethylstilbesterol in the ERα LBD were almost exactly the same as the known crystal structures of ERα in complex with these two estrogens. Using the same docking approach, we then characterized the binding interactions of 27 structurally similar E2 derivatives with the LBDs of human ERα and ERβ. While the binding modes of these E2 derivatives are very similar to that of E2, there are distinct subtle differences, and these small differences contribute importantly to their differential binding affinities for ERs. In the case of A-ring estrogen derivatives, there is a strong inverse relationship between the length of the hydrogen bonds formed with ERs and their binding affinity. We found that a better correlation between the computed binding energy values and the experimentally determined logRBA values could be achieved for various A-ring derivatives by re-adjusting the relative weights of the van der Waals interaction energy and the Coulomb interaction energy in computing the overall binding energy values.

  13. Compost mixture influence of interactive physical parameters on microbial kinetics and substrate fractionation.

    Science.gov (United States)

    Mohajer, Ardavan; Tremier, Anne; Barrington, Suzelle; Teglia, Cecile

    2010-01-01

    Composting is a feasible biological treatment for the recycling of wastewater sludge as a soil amendment. The process can be optimized by selecting an initial compost recipe with physical properties that enhance microbial activity. The present study measured the microbial O(2) uptake rate (OUR) in 16 sludge and wood residue mixtures to estimate the kinetics parameters of maximum growth rate mu(m) and rate of organic matter hydrolysis K(h), as well as the initial biodegradable organic matter fractions present. The starting mixtures consisted of a wide range of moisture content (MC), waste to bulking agent (BA) ratio (W/BA ratio) and BA particle size, which were placed in a laboratory respirometry apparatus to measure their OUR over 4 weeks. A microbial model based on the activated sludge process was used to calculate the kinetic parameters and was found to adequately reproduced OUR curves over time, except for the lag phase and peak OUR, which was not represented and generally over-estimated, respectively. The maximum growth rate mu(m), was found to have a quadratic relationship with MC and a negative association with BA particle size. As a result, increasing MC up to 50% and using a smaller BA particle size of 8-12 mm was seen to maximize mu(m). The rate of hydrolysis K(h) was found to have a linear association with both MC and BA particle size. The model also estimated the initial readily biodegradable organic matter fraction, MB(0), and the slower biodegradable matter requiring hydrolysis, MH(0). The sum of MB(0) and MH(0) was associated with MC, W/BA ratio and the interaction between these two parameters, suggesting that O(2) availability was a key factor in determining the value of these two fractions. The study reinforced the idea that optimization of the physical characteristics of a compost mixture requires a holistic approach. 2010 Elsevier Ltd. All rights reserved.

  14. Multi-spectroscopic studies on the interaction of human serum albumin with astilbin: Binding characteristics and structural analysis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jin; Li, Shuang; Peng, Xialian; Yu, Qing [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Department Chemistry and Chemical Engineering, Guangxi Normal University, Ministry of Education of China, Guilin 541004 (China); Bian, Hedong, E-mail: gxnuchem312@yahoo.com.cn [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Department Chemistry and Chemical Engineering, Guangxi Normal University, Ministry of Education of China, Guilin 541004 (China); Huang, Fuping [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Department Chemistry and Chemical Engineering, Guangxi Normal University, Ministry of Education of China, Guilin 541004 (China); Liang, Hong, E-mail: lianghongby@yahoo.com.cn [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Department Chemistry and Chemical Engineering, Guangxi Normal University, Ministry of Education of China, Guilin 541004 (China)

    2013-04-15

    Five spectroscopic techniques were used to investigate the interaction of astilbin (ASN) with human serum albumin (HSA). UV–vis absorption measurements prove that ASN–HSA complex can be formed. The analysis of fluorescence spectra reveal that in the presence of ASN, quenching mechanism of HSA is considered as static quenching. The quenching rate constant k{sub q}, K{sub SV} and the binding constant K were estimated. According to the van't Hoff equation, the thermodynamic parameters enthalpy change (ΔΗ) and entropy change (ΔS) were calculated to be −12.94 kJ mol{sup −1} and 35.92 J mol{sup −1} K{sup −1}, respectively. These indicate that the hydrophobic interaction is the major forces between ASN and HSA, but the hydrogen bond interaction cannot be excluded. The changes in the secondary structure of HSA which was induced by ASN were determined by circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy. -- Graphical abstract: In this paper, the interaction of HSA with ASN was systematically studied under simulated physiological conditions by using UV–vis absorption, CD, FT-IR, fluorescence and Raman spectroscopic approaches. The quenching constant k{sub q}, K{sub SV} and the binding constant K were estimated. The changes in the secondary structure of HSA were studied by Circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy. The UV–visible absorption spectra of HSA in the absence and presence of different concentration of ASN (1) and fluorescence spectra of HSA in the absence and the presence of ASN (2). Highlights: ► Interaction of ASN and HSA has been studied by five spectroscopic techniques. ► Hydrophobic interaction is the major forces between ASN and HSA. ► Binding of ASN induced the changes in the secondary structure of HSA.

  15. Na+/substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-labeled Substrate

    Science.gov (United States)

    Steed, P. Ryan; Stein, Richard A.; Mishra, Smriti; Goodman, Michael C.; Mchaourab, Hassane S.

    2013-01-01

    NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal to characterize mechanistically relevant binding interactions with NorM and to directly address the coupling of ion and drug binding. Fluorescence and EPR experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron-electron resonance (DEER) spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+ gradient across the native membrane, govern Na+/drug coupling during antiport. PMID:23902581

  16. Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model.

    Science.gov (United States)

    Sasaki, Takashi; Mita, Moeko; Ikari, Naho; Kuboyama, Ayane; Hashimoto, Shuzo; Kaneko, Tatsuya; Ishiguro, Masaji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-01-01

    TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5-nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse-human chimeric TGR5, we also found that three amino acid residues (Q77ECL1, R80ECL1, and Y893.29) are important in the hTGR5-nomilin