Dissertations / Theses on the topic 'Metal binding sites'
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Gapian, Bianké Jean-Paul. "DNA mimics containing artificial metal-binding sites /." [S.l.] : [s.n.], 2006. http://www.zb.unibe.ch/download/eldiss/06bianke_g.pdf.
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Torrance, James William. "The geometry and evolution of catalytic sites and metal binding sites." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612125.
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Higgins, Sean. "The design of conducting polymers with metal binding sites." Thesis, University of St Andrews, 1998. http://hdl.handle.net/10023/14786.
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This study is concerned with the synthesis of conjugated polyheterocycles with potential metal binding sites for applications in sensors, catalysis and electronics. The first synthetic approach to polyheterocycles was based on the interfacial polycondensation of a dihydrazide derivative of pyridine with a diacid chloride to produce a precursor polymer. It was shown, however, the starting materials could not be easily prepared in high yield. Model studies confirmed the feasibility of the route but these studies also suggested that the precursor polymers were unlikely to be very soluble. The second precursor route explored began with the preparation of 2,6-diethynylpyridine. The intermolecular Glaser coupling of the ethyne groups afforded the precursor polymer, poly((2,6'-pyridyl)but-l,3-diyne), as a black powder which was insufficiently soluble to allow conversion to the poly heterocycles. A series of dimers and trimers containing various combinations of 2-furyl, 2-thienyl and 2-pyridyl moieties were prepared using two different coupling procedures that yielded compounds with the required 2,2'-heteroatom arrangement as required for metal binding. Some of these monomers were electropolymerised and the metal binding properties of these polymers was investigated by cychc voltammetry. In particular, the two trimers: 2,5-di-(2-thienyl) pyridine; and 2,6-di-(2-thienyl) pyridine showed potential metal coordination despite their hydrophobic nature and impermeability towards metal complexes. Evidence was presented to suggest that these polymers are protonated during the electropolymerisation reaction. X-ray analysis of the 2,5-di-(2-thienyl) pyridine showed that only the 2,2'-linked thiophene was coplanar with the pyridine due to a charge transfer interaction. This interaction insures that S and N atoms have a planar syn arrangement conducive to metal binding. Several oligothiophenes were prepared to investigate methods for enhancing the solubility of polyheterocycles. The knowledge gained from these investigations was used to prepare a series of regiochemically well-defined poly((3-alkyl)thiophenes). The regularity of these polymers was confirmed by NMR analysis. Related monomers were prepared containing the necessary solubilising alkyl groups as well as phenyl groups designed to act as acceptor ligands for the low-valent transition metals such as ruthenium(II). The electrochemistry of these novel thiophene monomers is reported.
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Gregory, David St John. "Prediction, design and characterisation of metal binding sites in antibodies." Thesis, University of Oxford, 1992. https://ora.ox.ac.uk/objects/uuid:630ede76-2db6-4e59-bdbe-d337d4fe07a9.
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The design and creation of a functional metalloanti body is presented. A survey of metalloprotein structures was carried out to determine the common structural features of metal binding sites. Metal ligands were then introduced into hypervariable loop 11 of the anti-lysozyme antibody HyHEL-5 and the site comprehensively modelled. The final model was used as a basis for site-directed mutagenesis and mutant antibodies were produced using a Xenopus laevis oocyte expression system. Antigen binding studies showed the mutants to have an affinity for lysozyme equivalent to the parent antibody, while subsequent metal binding experiments have shown the new site to be capable of binding the transition metals cobalt, nickel, copper, zinc and cadmium. A method for the rapid prediction and design of metal binding sites in proteins is described. A protein structure is screened for the location of putative metal sites by template matching. After the introduction of suitable liganding residues, the metal binding potential at each site is evaluated using hydrophobicity contrast. A point representing the metal ion within a new site may be optimised with-respect-to the ligands using multidimensional minimisation. The method has been tested on known metalloprotein structures. It was routinely able to identify the metal binding sites and position a metal point in the site to within 0.6A of the actual metal ion.
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Hannan, Jonathan Paul. "NMR spectroscopic studies of transition metal binding sites in metalloproteins." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323296.
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Uppal, Daljit Kaur. "Studies of the metal-binding sites in macrocyclic quadridentate ligands." Thesis, London Metropolitan University, 1986. http://repository.londonmet.ac.uk/3284/.
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Jeong, Chang-Yoon. "Modelling metal competition for adsorption sites on humic acid." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389363.
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McMahon, Jennifer Nicole. "Heavy metal competition for acid volatile sulfide binding sites in southeastern coastal sediments." Thesis, Georgia Institute of Technology, 1997. http://hdl.handle.net/1853/19134.
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Yang, Ying. "Mechanism of metal delivery and binding to transport sites of Cu+-transporting ATPases." Link to electronic thesis, 2005. http://www.wpi.edu/Pubs/ETD/Available/etd-042905-112044/.
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Lazarides, Theodore. "Luminescent d-block metal polypyridyl complexes bearing secondary macrocyclic or non-macrocyclic binding sites." Thesis, University of Sheffield, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427184.
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Chandrasekar, Sowmya. "Probing metal and substrate binding to metallo-[beta]-lactamase ImiS from Aeromonas sobria using site-directed mutagenesis." Oxford, Ohio : Miami University, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1098134311.
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Keech, Angus Miles. "Role of cobalt(II) and manganese(II) as optical and magnetic probes of metal binding sites in proteins." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389267.
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Prasannan, Charulata Bhaskaran. "Modulation of restriction enzyme PvuII activity by metal ion cofactors." Diss., St. Louis, Mo. : University of Missouri--St. Louis, 2009. http://etd.umsl.edu/r4461.
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Ow, Yang Giselle Bei. "EXAFS structural analysis on metal binding sites in wood pulp and a brief study on chelation of metals in bleaching." Diss., Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/7087.
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Haas, Marco. "A synthetic approach to asymmetric phthalocyanines with peripheral metal-binding sites and their divalent Ruthenium complexes /." [S.l.] : [s.n.], 2006. http://www.zb.unibe.ch/download/eldiss/06haas_m.pdf.
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Soebbing, Samantha Lynn. "Incorporation of histidine-rich metal-binding sites onto small protein scaffolds implications for imaging, therapeutics, and catalysis /." Diss., University of Iowa, 2008. http://ir.uiowa.edu/etd/37.
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Schweitzer, Dirk. "Biomimetic models of the active site of the metalloenzyme nitrile hydratase /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8692.
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Wenzel, Nathan F. "Glyoxalase 2-2 over-expression and characterization of a metallohydrolase from Arabidopsis thaliana /." Oxford, Ohio : Miami University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1069728340.
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Thesis (M.S.)--Miami University, Dept. of Chemistry and Biochemistry, 2003.Title from first page of PDF document. Document formatted into pages; contains xii, 83 p. : ill. Includes bibliographical references.
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Roy, Choudhury Shamali. "Mapping of metal ion binding sites in the RecBCD enzyme and the role of magnesium in the subunit interactions of RecBCD." College Park, Md. : University of Maryland, 2006. http://hdl.handle.net/1903/3360.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2006.Thesis research directed by: Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Rehmani, Imran J. "Studying the DNA Binding and Conformation of Metal-Binding Site Mutations in Pirin." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/chemistry_theses/53.
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The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.
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Eyley, J. E. "Gas sorption and binding site studies in metal organic frameworks." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/40546/.
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This thesis describes the design and synthesis of a series of Cu(II)-paddlewheel based metal organic frameworks (MOFs) for the adsorption of gaseous fuels and pollutants. The frameworks comprise V-shaped pyridyl carboxylate ligands, which are progressively modified to increase gas adsorption capacity and selectivity of the resultant materials. Chapter 1 introduces the history and structure of MOFs, including an exploration of their varied applications. Particular attention is paid to gas adsorption within these materials, considering the best performing materials for each gas discussed. Chapter 2 explores the ability of uncoordinated pyridyl groups to form strong interactions with adsorbed CO2. A new self-interpenetrated Cu(II) MOF (MFM-170) is synthesised from a V-shaped pyridyl carboxylate ligand, in which the pyridyl nitrogen coordinates to the axial site of the interpenetrating net. The porosity and gas adsorption properties of this material are discussed in detail. Chapter 3 describes how functional groups can introduce binding sites into MOFs, strengthening framework-adsorbate interactions and improving gas adsorption capacities. Three isostructural analogues of MFM-170 are synthesised (MFM-171, MFM-172, MFM-173), each with a different functional group directed into the pore. Differences in the gas adsorption properties of these materials are rationalised by identification of CO2 binding sites by IR microspectroscopy and Single Crystal X-ray Diffraction experiments. Chapter 4 draws on knowledge of CO2 binding sites identified in Chapter 3 to selectively target areas of the framework where strongly coordinating functional groups would have the greatest effect of CO2 adsorption and selectivity. A new Cu(II) framework (MFM-175) is reported, incorporating triazole groups directed into the void. The structure, stability and gas adsorption properties of MFM-175 are studied in detail and compared to MFM-170. Chapter 5 investigates the synthesis of a non-interpenetrated analogue of MFM-170 with the aim of liberating the pyridyl nitrogen group to improve framework-adsorbate interactions. A new Cu(II) MOF (MFM-176) is synthesised, featuring a two-fold interpenetrated structure. Nevertheless MFM-176 demonstrates improved selectivity parameters and additionally a promising strategy for the successful synthesis of a non-interpenetrated framework.
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Mongeau, Raymond. "Antidepressant and anxiolytic action on the Serotonin1A binding site." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59934.
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Several lines of evidence suggest an involvement of serotonin$ sb{ rm 1A}$ (5-HT$ sb{ rm 1A}$ receptors in the regulation of emotions. In order to investigate the molecular basis of recent electrophysiological findings which implicated 5-HT$ sb{ rm 1A}$ receptors in the mechanism of action of antidepressants and anxiolytics, radioligand binding and autoradiographic studies using tritiated 8-hydroxy-2-(di-N-propylamino)-tetralin ( ($ sp3$H) -8-OH-DPAT) were done in rat brain following various treatments. These included: the tricyclic antidepressant imipramine; the reuptake blockers paroxetine and indalpine; the monoamine oxidase inhibitor clorgyline; electroconvulsive shock; lithium; the classic benzodiazepine diazepam; and the 5-HT$ sb{ rm 1A}$ partial agonist gepirone. None of these treatments, nor the fluctuation in 5-HT availability provoked by the circadian cycle, gave any significant changes, with the exception of clorgyline which initially appeared to decrease the affinity of ($ sp3$H) -8-OH-DPAT for its receptor. A further series of studies in vitro and in vivo ascertained the possibility that the 5-HT$ sb{ rm 1A}$ receptors may display two interconvertible affinity states and that, in fact, clorgyline induces a shift of the high to the lower affinity state. The findings from this second series of experiments suggested that labile changes, which may possibly be disrupted during membrane preparation, in the coupling between the 5-HT$ sb{ rm 1A}$ receptor and a guanine nucleotide binding protein (G-protein) may account for the effects that certain treatments have on 5-HT$ sb{ rm 1A}$ receptor responsiveness.
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Lee, Hsiau-Wei. "Determining The Site Specific Metal Binding and Structural Properties of EF-Hand Protein Using Grafting Approach." Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/chemistry_diss/26.
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Calmodulin is an essential EF-hand protein with a helix-loop-helix calcium binding motif. Understanding Ca(II) dependent activation of calmodulin and other EF-hand proteins is limited by Ca(II)-induced conformational change, multiple and cooperative binding of Ca(II) ions, and interactions between the paired EF-hand motifs. The goal of this research project is to probe key determinants for calcium binding properties and pairing interactions at the site specific level using a grafting approach and high resolution NMR. An individual Ca(II) binding site of the EF-hand motifs of calmodulin was grafted into a non-calcium dependent protein, CD2, to bypass limitations associated with natural EF-hand proteins and peptide fragments. Using high resolution NMR, we have shown that the grafted EF-loop III of calmodulin in the host protein retains its native conformation with a strong loop and β-conformation preference. Grafted ligand residues in the engineered protein are directly involved in binding of Ca(II) and La(III). The NMR studies support our hypothesis that both ligand arrangement and dynamic properties play essential role in tuning Ca(II) binding affinities. Using pulse-field diffusion NMR and protein engineering, we further demonstrated that grafted EF- loop remains as a monomer. Although the EF-loop with flanking helices dimerizes in the presence of Ca(II). Additionally, removal of conserved hydrophobic residues at the flanking helices of the EF-hand motif leads to be monomer in the absence and presence of metal ions. Our results suggest that conserved hydrophobic residues are essential for the pair-paired interaction in the coupled EF-hand protein. We have shown that our developed grafting approach can be applied to probe intrinsic Ca(II) binding affinities of different Ca(II) binding sites.
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Luo, Yujia. "Analgesic Effect of Nicotine and Exploration of Binding Sites for α4β2 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators." Thesis, The University of Sydney, 2023. https://hdl.handle.net/2123/30000.
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Pain is a leading cause of the global burden of disease and disability. Nicotinic acetylcholine receptors (nAChRs), especially the α4β2 subtype, are widely expressed throughout the central nervous system (CNS) and peripheral nervous system (PNS), making them critical analgesic targets for the development of novel drugs. In the early part of this thesis, the analgesic effects of nicotine were quantified through meta-analysis. Sixteen eligible articles, including five studies on pain tolerance (n = 210), five studies on pain threshold (n = 210), and 11 studies on pain scores (n = 1,148) were included. Prolonged laboratory-induced pain threshold and tolerance time and mild postoperative pain relief were observed in patients who received acute nicotine therapy. In addition, a network meta-analysis was conducted to compare the effectiveness of nicotine transdermal patch and nasal spray for postoperative pain. Eight eligible studies (n = 555) were included. The results showed that the nicotine transdermal patch had a stronger analgesic effect than the nicotine nasal spray did. In addition, the analgesic effect of the nicotine transdermal patch peaked at 1-1.5 hours. The results of our analysis revealed that nicotine produces a rapid analgesic effect in humans, but the long-term use of nicotine is likely to cause hyperalgesia. The side effects of nicotine have been reported in several studies, likely due to the absence of nAChR subtype selectivity of nicotine. This limits the clinical applications of nicotine. Consequently, there is a need to study nAChR-targeted compounds with high receptor selectivity in order to replace nicotine.
Pharmacological studies of subtype-selective positive allosteric modulators (PAMs) with regard to the α4β2 nAChR (the most abundant subtype of nAChR in humans) are described in the following part of this thesis. The α4β2 nAChR is the most highly expressed subtype of nAChRs in the human brain, where it forms a high-affinity binding site for nicotine. NS206 was identified as a selective PAM of α4β2 nAChRs. LY2087101 and dFBr have pharmacological profiles similar to those of NS206, suggesting that they may bind to the same binding site(s) in wild-type (α4)3(β2)2 nAChRs. Notably, the α4 and β2 subunits assembled into a mixture of (α4)2(β2)3 (high-affinity) and (α4)3(β2)2 (low-affinity) nAChRs in Xenopus laevis oocytes, and the two stoichiometries differ significantly in their functional and pharmacological properties, for instance, the (α4)3(β2)2 nAChR exhibits a biphasic ACh concentration-response relationship, whereas the (α4)2(β2)3 nAChR exhibits a monophasic ACh concentration-response relationship. Therefore, the pharmacological studies on NS206 have been conducted on both stoichiometries. A uniform population of either (α4)3(β2)2 or (α4)2(β2)3 nAChRs in oocytes was obtained by injecting with α4 and β2 cRNA at ratios of 10:1 and 1:4, respectively. The addition of NS206 increased the height of peak responses at (α4)3(β2)2 and (α4)2(β2)3 nAChRs (2.04 ± 0.64 vs. 0.88 ± 0.13, and 2.61 ± 0.42 vs. 0.96 ± 0.03, respectively), which is a statistically significant difference. However, a change in EC50s of the ACh concentration-response curves was not observed. In addition, no significant additive effect or change in potency was observed when comparing the NS206 + LY2087101 and NS206 + dFBr groups to the NS206, LY2087101 or dFBr groups.
To determine the binding sites of NS206, amino acid substitutions of phenylalanine-substituted leucine at position 256 (α4L256F) and leucine-substituted phenylalanine at position 316 (α4F316L) in the transmembrane domain (TMD) of the α4-subunit were performed using site-directed mutagenesis technique. Compared with the wild-type (α4)3(β2)2 and (α4)2(β2)3 nAChRs, the mutated receptors with amino acid substitutions α4L256F and α4F316L significantly reduced NS206 potentiated maximum responses of ACh-evoked currents [(α4)3(β2)2: 6.91 ± 0.57 vs. 6.23 ± 4.35 and 0.84 ± 1.18, respectively; (α4)2(β2)3: 4.14 ± 0.38 vs. 2.37 ± 0.1 and 0.94 ± 0.14, respectively]. In addition, the NS206 potency showed a right-shifted trend for the mutated nAChRs. These results indicate that α4L256 and α4F316 are potential binding sites for NS206 in α4β2 nAChRs.
In summary, this thesis first discussed the involvement of nAChRs in pain by determining the analgesic effect of nicotine on laboratory-induced pain and acute postoperative pain. Furthermore, we compared the analgesic effects of two common clinical nicotine dosage forms, the nicotine transdermal patch and nasal spray, and determined the time of the peak analgesic effect. We found that the analgesic effect of the nicotine transdermal patch was better than that of nicotine nasal spray, peaking at approximately 1-1.5 hours. This thesis studied the pharmacological profiles of NS206 and investigated the binding interactions of NS206, LY2087101, and dFBr. We found that adding NS206 hardly changed the EC50s of ACh concentration-response curves, but significantly increased the height of peak responses at (α4)3(β2)2 and (α4)2(β2)3 nAChRs. Furthermore, no significant additive effect or change in potency was observed when comparing the NS206 + LY2087101 and NS206 + dFBr groups to the NS206, LY2087101 or dFBr groups. Finally, we identified two potential binding sites of NS206. Compared to the wild-type (α4)3(β2)2 and (α4)2(β2)3 nAChRs, the mutated receptors with amino acid substitutions α4L256F and α4F316L significantly reduced NS206 potentiated maximum responses of ACh-evoked currents. Furthermore, the NS206 potency showed a right-shifted trend for mutated nAChRs. These results indicate that α4L256 and α4F316 are the potential binding sites of NS206 on the α4β2 nAChRs. Combined with molecular modelling these results may facilitate delineation of the binding mode of the studied compounds and may thereby pave the way for the future rational design and discovery of novel modulators.
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Chandrasekar, Sowmya. "Probing Metal and Substrate Binding to Metallo-β-Lactamase ImiS from Aeromonas Sobria using Site-Directed Mutagenesis." Miami University / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=miami1098134311.
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Zhao, Kun. "Mathematical Methods for Network Analysis, Proteomics and Disease Prevention." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/math_diss/6.
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This dissertation aims at analyzing complex problems arising in the context of dynamical networks, proteomics, and disease prevention. First, a new graph-based method for proving global stability of synchronization in directed dynamical networks is developed. This method utilizes stability and graph theories to clarify the interplay between individual oscillator dynamics and network topology. Secondly, a graph-theoretical algorithm is proposed to predict Ca2+-binding site in proteins. The new algorithm enables us to identify previously-unknown Ca2+-binding sites, and deepens our understanding towards disease-related Ca2+-binding proteins at a molecular level. Finally, an optimization model and algorithm to solve a disease prevention problem are described at the population level. The new resource allocation model is designed to assist clinical managers to make decisions on identifying at-risk population groups, as well as selecting a screening and treatment strategy for chlamydia and gonorrhea patients under a fixed budget. The resource allocation model and algorithm can have a significant impact on real treatment strategy issues.
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Chen, Yinghua. "Solution structure of the target recognition domain of zoocin A, an antibacterial enzyme, and the metal binding site of zoocin A." Thesis, [Tuscaloosa, Ala. : University of Alabama Libraries], 2009. http://purl.lib.ua.edu/2202.
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Kreibich, Julian. "Mechanistic insights into carbon monoxide and CoA binding at the Ni,Ni-[4Fe-4S] active site of the acetyl-CoA synthase from Carboxydothermus hydrogenoformans." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/23215.
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Die Acetyl-CoA Synthase (ACS) beinhaltet ein einzigartiges Metallcluster (Cluster A) in seinem aktiven Zentrum, welches wichtig für ein autotrophes Wachstum von Bakterien und Archaeen ist, die den reduktiven Acetyl-CoA-Weg nutzen. Der letzte Schritt dieses Syntheseweges wird von der ACS am proximal zum [4Fe-4S] Cluster liegenden Ni ion katalysiert (Nip). In meiner Arbeit wurde die ACS von Carboxydothermus hydrogenoformans (ACSCh) auf seine Ligandenbindung untersucht und in verschiedenen Konformationen kristallisiert.
Zuerst wurde die ACSCh in einer neuen Konformation (ACSCh-closed) kristallisiert und deren Struktur mit einer Auflösung von 2.1 Å bestimmt. Diese wies einen geringeren Kontakt zur Solventumgebung auf als die vorher bekannte Struktur der ACSCh (ACSCh-open, PDB-ID: 1RU3). Das Cluster A der ACSCh-closed unterscheidet sich von der ACSCh-open in der Koordination des Nip, welches verzerrt tetraedrisch koordiniert in ACSCh-closed vorliegt und quadratisch planar in ACSCh-open. Eine Analyse des Modells wies einen molekularen Tunnel auf, der nur in ACSCh-closed vorhanden ist, welcher als CO-Kanal zur Substratversorgung dienen könnte.
Zweitens wurde die Kristallstruktur einer CO gebunden ACSCh-closed mit einer Auflösung von 2.0 Å gelöst. Darin wurde eine Elektronendichte am Nip identifiziert, die als CO überzeugend modelliert werden konnte. CO bindet am ebenfalls verzerrt tetraedrisch koordinierten Nip. Die Konformationsänderung von ACSCh-open zu ACSCh-closed scheint der entscheidende Schritt zu sein, um CO zur Bindungsstelle zu leiten.
Die dritte Struktur zeigt eine CoA gebundene ACSCh Struktur mit einer Auflösung von 2.3 Å. CoA bindet am Nip, wobei Nip quadratisch planar koordiniert wird. Die Gegenwart von CoA wurde mit Berechnungen verschiedener Elektronendichte-Karten für CoA validiert.
Die Bindung von CO und CoA an ACSCh wurde zudem mittels isothermaler Titrationskalorimetrie weiter charakterisiert. Dabei bindet CoA enthalpisch getrieben mit einem KD von 3.1 µM und CO entropisch getrieben mit einem KD von 9.4 µM an ACSCh.The acetyl-CoA synthase (ACS) harbors a unique metal cluster (cluster A) in its active site, which is important for bacteria and archaea to survive in autotrophic growth using the reductive acetyl-CoA. The last step of this pathway is catalyzed by ACS at the Nip, the Ni ion proximal to the [4Fe-4S] cluster. In my study, the monomeric ACS of Carboxydothermus hydrogenoformans (ACSCh) was studied in its ligand binding and crystallized in different forms. At first, an ACSCh structure was solved in a new conformation at dmin of 2.1 Å and less solvent exposed (called ACSCh-closed) than the known structure of ACSCh (called ACSCh-open, PDB-ID:1RU3). The cluster A of ACSCh-closed differs to that of ACSCh-open in the coordination of the proximal Ni, which is distorted tetrahedrally coordinated in ACSCh-closed and square planar coordinated in ACSCh-open. Analysis of the model revealed a molecular tunnel that is only present in ACSCh-closed, which might act as CO channel for substrate delivery. Secondly, a CO-bound ACSCh-closed crystal was obtained and solved at a resolution of 2.0 Å. An electron density fitting with a diatomic ligand at the Nip site was clearly identified and modeled as a CO molecule. CO binds at the Nip site completing the tetrahedral coordination geometry of Nip. The conformational switch between open and closed is responsible for CO migration and binding to the catalytic site. A third crystal structure depicts a CoA bound ACSCh structure at 2.3 Å resolution. CoA binds at the Nip which shows a square planar geometry. This was further supported by calculating different electron density maps for CoA. The binding of CO and CoA to ACSCh has been characterized by isothermal calorimetry experiments. While CoA binding is enthalpically driven with a KD of 3.1 µM, CO binds to ACSCh by entropic contribution with a KD of 9.4 µM.
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Houwaart, Torsten. "Cobalt porphyrins on coinage metal surfaces - adsorption and template properties." Thesis, Lyon, École normale supérieure, 2014. http://www.theses.fr/2014ENSL0927.
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Cette thèse est une étude théorique sur la interface de porphyrine de cobalt avec des surfaces métalliques avec le code VASP DFT. Le cadre DFT nécessaire a été introduit dans le chapitre 1. La structure de la jBardeen, une programme ecrit en Java, pour la simulation de la STM est expliqué dans le chapitre 2 et le code source est jointe en annexe. Une étude de l'adsorption de CoTPP sur les surfaces métalliques a été entrepris dans le chapitre 3. Différents paramètres de calcul ont été évalués: Le site d'adsorption et de la géométrie à la fois la molécule et la surface ont été étudiés par rapport à la xc-fonctionnel et correction de la dispersion utilisée. Une adsorption site le plus stable est identifié. Par conséquent, ce site plus stable a été étudiée pour sa structure électronique. Calculés images STM avec le code jBardeen ont été comparés avec une experimentation de CoTPP Cu sur une surface (111) avec une couverture sous monocouche. Dans le chapitre 4, un adatome Fe a été présenté à la CoTPP sur Ag système (111). Trois sites de liaison symétrique différentes pour l'atome Fe ont été identifiés sur le macrocycle, marqué les , bi-, brd- et bru-positions. Un moment magnétique pouvait être attestée qui a été principalement situé sur l'atome Fe. Voies possibles entre les quatre, symétriquement équivalentes, sites bi- ont été étudiées avec des méthodes différentes. Simples calculs dans le vacuum et calculs de la “Nudged Elastic Band” (NEB) de l'ensemble du système a révélé une hauteur de barrière légèrement au-dessus de 0,2 eV allant de position bi à la posititon brd. Une analyse de vibration a montré que la commutation de l'atome Fe est susceptible, lorsqu'il est perturbé hors d'équilibre dans les positions brd et bruThis thesis is a theoretical study on the cobalt porphyrin - coinage metal surface interface with the DFT code VASP. The necessary DFT framework has been introduced in chapter 1. The structure of the Java program jBardeen for STM simulation is explained in chapter 2 and the source code is attached as Appendix. A study of the adsorption of CoTPP on coinage metal surfaces has been undertaken in chapter 3. Different parameters of the calculation have been evaluated: the adsorption site and the geometry of both the molecule and surface have been investigated with respect to the xc-functional and dispersion correction used. A most stable adsorption site -bridge down- is identified. Consequently, this most stable site was investigated for its electronic structure. Calculated STM images with the jBardeen code were compared with an experiment of CoTPP on a Cu(111) surface with sub monolayer coverage. In chapter 4 an Fe adatom was introduced to the CoTPP on Ag(111) system. Three symmetrically different binding sites for the Fe atom were identified on the macrocycle, labelled the bi-, brd- and bru-positions for bisector, bridge down and bridge up respectively. A magnetic moment could be evidenced which was mainly located on the Fe atom. Possible pathways between the four symmetrically equivalent bisector sites were investigated with different methods. Single point calculations in vacuum and Nudged Elastic Band (NEB) of the whole system revealed a barrier height of slightly above 0.2 eV going from bi- to the brd-position. A vibrational analysis showed that switching of the Fe atom is likely, when perturbed out of equilibrium in the brd- and bru- positions
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López, Muñoz Laura. "Homology modeling and structural analysis of the antipsychotic drugs receptorome." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7228.
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Classically it was assumed that the compounds with therapeutic effect exert their action interacting with a single receptor. Nowadays it is widely recognized that the pharmacological effect of most drugs is more complex and involves a set of receptors, some associated to their positive effects and some others to the side effects and toxicity. Antipsychotic drugs are an example of effective compounds characterized by a complex pharmacological profile binding to several receptors (mainly G protein-coupled-receptors, GPCR). In this work we will present a detailed study of known antipsychotic drugs and the receptors potentially involved in their binding profile, in order to understand the molecular mechanisms of the antipsychotic pharmacologic effects.The study started with obtaining homology models for all the receptors putatively involved in the antipsychotic drugs receptorome, suitable for building consistent drug-receptor complexes. These complexes were structurally analyzed and compared using multivariate statistical methods, which in turn allowed the identification of the relationship between the pharmacological properties of the antipsychotic drugs and the structural differences in the receptor targets. The results can be exploited for the design of safer and more effective antipsychotic drugs with an optimum binding profile.
Tradicionalmente se asumía que los fármacos terapéuticamente efectivos actuaban interaccionando con un único receptor. Actualmente está ampliamente reconocido que el efecto farmacológico de la mayoría de los fármacos es más complejo y abarca a un conjunto de receptores, algunos asociados a los efectos terapéuticos y otros a los secundarios y toxicidad. Los fármacos antipsicóticos son un ejemplo de compuestos eficaces que se caracterizan por unirse a varios receptores simultáneamente (principalmente a receptores unidos a proteína G, GPCR). El trabajo de la presente tesis se ha centrado en el estudio de los mecanismos moleculares que determinan el perfil de afinidad de unión por múltiples receptores de los fármacos antipsicóticos.
En primer lugar se construyeron modelos de homología para todos los receptores potencialmente implicados en la actividad farmacológica de dichos fármacos, usando una metodología adecuada para construir complejos fármaco-receptor consistentes. La estructura de estos complejos fue analizada y se llevó a cabo una comparación mediante métodos estadísticos multivariantes, que permitió la identificación de asociaciones entre la actividad farmacológica de los fármacos antipsicóticos y diferencias estructurales de los receptores diana. Los resultados obtenidos tienen interés para ser explotados en el diseño de fármacos antipsicóticos con un perfil farmacológico óptimo, más seguros y eficaces.
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Lin, Yu-Feng, and 林玉鳳. "Prediction of metal binding sites in proteins." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/86030438365561006913.
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碩士中國醫藥大學
分子系統生物醫學研究所
98
The biochemical functions of proteins are determined upon the structure of the polypeptide and the interaction of cofactors. The functional surfaces of proteins, which interact with ligands, substrates, or proteins, tend to be more conserved in sequence and structure pattern, and the residues of a functional binding region are usually spatial close in three-dimensional structure. Recently, there are more than sixty thousand structures in Protein Data Bank (PDB), and a large amount of proteins, about one-third of proteins, is considered binding to metal ions. Large parts of metal ions in human body are bound to proteins, and metal ions are very important to implement the functions of metalloproteins. However, the subsequent experimental method to localize metal binding sites is time-consuming and costly, so that many computational methods had been developed for indentifying metal binding sites. Most computational methods are based on sequence information; therefore, we proceeded to our investigation of metal binding sites by using sequence and structural information. In this study, six kinds of metal ions (Calcium, Copper, Iron, Magnesium, Manganese, Zinc) binding residues had been constructed, and then gathered as metal-binding residue templates, which are metal binding residues spherical within 3.5Å around the site of interest. By taking advantage of the fragment transformation method, the comparison of structures was carrying out. According to the sequence and structure conservation of interaction sites, we can combine both structural and sequence information to identify the local structure protein-metal interaction sites. In the results, the prediction performance of a 94.6% Q2 accuracy with a 60.5% TPR at a 5% FPR can be achieved.
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Li, Jyn-Han, and 李居翰. "MIB: a Metal Ion–Binding sites prediction tool." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/8rn739.
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Yang, Tzung Yin, and 楊宗穎. "Mononuclear versus Binuclear Metal-Binding Sites: Metal Binding Affinity and Selectivity from PDB Survey and DFT/CDM Calculations." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/09710750216292499288.
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碩士國立清華大學
化學系
95
Binuclear metal centers in metalloenzymes are involved in a number of hydrolytic, hydration, isomerization, and redox processes. Despite the growing number of studies elucidating their structure, properties, and function, questions regarding certain aspects of the bimetallic proteins’ biochemistry still remain; e.g., (i) What are the general characteristics of binuclear sites found in 3D structures such as the range of metal訃metal distances, and the most common ligand bridging the two metal cations? (ii) How does the presence of a metal cation in one of the binuclear sites affect the metal訃binding affinity/selectivity of the other site? (iii) How do the characteristics and metal訃binding affinity/selectivity of binuclear sites compare with those of their mononuclear counterparts? Here we address these questions by combining a Protein Data Bank survey of binuclear sites with density functional theory (DFT) combined with continuum dielectric method (CDM) calculations. The results reveal that for homo訃binuclear sites, the metal separation depends on the metal’s charge and the electron-accepting ability, and Asp-/Glu-, bidentately bound to the two cations, is the most common bridging ligand. They also reveal that Mg2+洶occupying one of the binuclear sites attenuates the metal訃binding affinity, but enhances the selectivity of its neighboring site, compared to the corresponding mononuclear counterparts. These findings are consistent with available experimental data. The weak metal binding of one of the binuclear sites would enhance the metal cofactor mobility in achieving the transition state, whereas the enhanced selectivity of Mg2+訃Mg2+ centers help protect against unwanted substitutions by transition metal ions, which are generally stronger Lewis acids compared to Mg2+.
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VALASATAVA, YANA. "NEW COMPUTATIONAL APPROACHES TO THE STUDY OF METALS IN BIOLOGY." Doctoral thesis, 2015. http://hdl.handle.net/2158/998429.
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Mazmanian, Karine, and 馬玫霓. "Elucidating the Physicochemical Principles of Regulation in Protein Functional Sites: Free Cysteines and Metal-binding Sites." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/s2d777.
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博士國立臺灣大學
生化科學研究所
106
Protein activity in the cell is tightly regulated on both cellular and molecular levels. Molecular level involves regulation through creating a local protein environment around a functional site organized for an optimal protein activity. Currently, general physicochemical principles of regulation of local environment of functional sites in proteins remain elusive and in this study, we tried to answer the question of how can interactions of the functionally important residues favor the optimal organization of the local protein environment. As examples of such functional systems, we have chosen free cysteines, zinc and sodium-binding proteins. To address the posed question we modeled the studied functional systems together with their interactions explicitly and implicit solvent with the quantum chemical density functional (QC/DFT) approach. The computational results were complemented with analysis and survey of the available experimental data. Cysteine (Cys) reactivity depends on the protein environment and one of the key contributors to its modulation is the HB interactions. Thus, we assessed the most preferred HB partners of free Cys. As a result, we show that the strength and selectivity of the HB interactions with free Cys ultimately depend on the combination of the extent of HB complex solvation and the properties of Cys HB partner. In this manner, through providing various HBs partners and dielectric environment the protein of interest can modulate the HB strength and consequently the degree of thiolate stabilization, which in turn would affect the cysteine''s nucleophilicity/reactivity. Statistical analysis of free Cys and their local protein environment in the reported crystal structures revealed that in the proteins the HB interactions with Cys are formed according to our predicted HB preferences. In proteins, cysteines are preferred first-shell ligands for zinc (Zn2+), and zinc is readily displaced by toxic cadmium (Cd2+) due to the high affinity of the later for cysteines. Proteins through providing HB interactions between the first- and second-shell ligands regulate and stabilize/protect the metal complex or enhance metal binding properties. Yet, knowledge of the preferred HB partners of metal ligands in different metalloproteins is lacking. We were the first to systematically unravel the principles governing the preferred hydrogen-bonding partners of metal ligands on the example of zinc- and cadmium-thiolate (Cys-) complexes in proteins. It appears that the nature of metal-binding first-shell residues and solvent exposure of the complex determine the HB preferences of the second shell ligands. Upon Zn2+ to Cd2+ substitution, the size difference of the two cations yielded similarities and differences in their protein environment preferences. We conclude that neutral and flexible Zn2+-sites lined by small neutral amino acid (aa) sidechains are more vulnerable for toxic Cd2+ effects as it could distort the native protein structure and thereby alter/abolish the protein function. Similarly to zinc proteins, sodium-binding proteins play diverse important roles in the cell. However the concentration of Na+ and Mg2+ cations are present in comparable amounts in the cytosol, but it is not clear how Na+ and Mg2+ binding sites discriminate the native cation among non-cognate ones from the surrounding milieu and the physical basis governing the selectivity for Na+ over Mg2+. The results, which are consistent with available experimental data, reveal that in proteins, the selectivity for Na+ over Mg2+ in sodium-binding sites stem mainly from the size, charge, and charge-accepting ability differences between Na+ and Mg2+. Next, we studied the competition between native Na+ and abiogenic Li+ in sodium neurotransporters and G-protein coupled receptors (GPCRs). Neurotransmitter transporters and receptors are well-known drug targets for psychiatric disorders and addictive behavior and lithium has been used as a first-line treatment of the number of psychiatric disorders (e.g. depression). We show that rigid Na+-sites that are crowded with multiple protein ligands are well-protected against Li+ attack, but their flexible counterparts or buried Na+-sites containing only one or two protein ligands are vulnerable to Li+ substitution. By displacing Na+ bound to an Asp- and a Ser in the solvent-inaccessible metal-binding sites of the A2AAR adenosine and the 1AR adrenergic GPCRs, Li+ could stabilize the receptor''s inactive state by prohibiting conformational changes to the active state, thus leading to decreased G-protein activity, which are hyperactive in bipolar patients. In our studied model systems, we have demonstrated how key physicochemical factors govern the formation of the optimal local environments around functional sites allowing proteins to regulate their functions. Our results can be broadly applied to artificial protein design, rational drug design, and development of biosensors. We underscore the importance of further investigations of physicochemical local environments of the functional sites and their role in the regulation of protein functions and outline several problems for future investigations.
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Switala, Brooke A. "Probing metal binding sites in DNA by using europium(III) luminescence spectroscopy." 2007. http://proquest.umi.com/pqdweb?did=1441197201&sid=1&Fmt=2&clientId=39334&RQT=309&VName=PQD.
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Thesis (M.A.)--State University of New York at Buffalo, 2007.Title from PDF title page (viewed on June 18, 2008) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Morrow, Janet R. Includes bibliographical references.
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Chen, Yu-Kai, and 陳又楷. "Analysis of Complementary Transgenic Arabidopsis and the Metal Binding Sites in Phytochelatin Synthase." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/47063052983945167425.
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Pai, Chi-Yi, and 白吉鎰. "Prediction Metal-Binding Sites of Metalloprotein using RBF network and physicochemical properties analysis." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/96410553233806878376.
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碩士元智大學
資訊工程學系
98
Metalloprotein is a Metal and protein binding type of people needed.Some of Metalloproteins that are become Zinc-finger structure,these are Transcription factor with DNA binding for the regulation of gene expression.This is a important regulation organisms,play an important role in biological process. In the experimental difficulties, a further analysis of its binding site, it is very important and useful work, so to develop a predictable and metal ions in protein binding site is an important study. In this experiment the use of QuickRBF classifier predicted protein sequences with the metal binding site, and to use sequence information and some predict the evolution of the physical and chemical properties to predict the protein sequence of cysteine and histidine . In addition also proposed disulfide bond forecasting methods and histidine forecasting methods used to improve QuickRBF predictions. Our forecast performance for cysteine can be achieved Recall 60.3%, Precision 88.8%, while the histidine can be achieved Recall 40.1%, Precision 62.3%
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Bridgewater, Juma D. "Using metal catalyzed oxidation reactions and mass spectrometry as a reliable method for determining metal binding-sites in proteins." 2006. https://scholarworks.umass.edu/dissertations/AAI3242308.
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The combination of metal catalyzed oxidation (MCO) reactions and mass spectrometry (MS) is a relatively new method of determining the coordination structure of metalloproteins. The sensitivity of the MCO/MS method gives it significant potential for the study of protein structure. The method takes advantage of Fenton-type oxidation reactions occurring at protein-bound transition-metal ions. These reactions produce reactive oxygen species that modify metal binding residues, and the modified residues are identified using the peptide sequencing ability of MS. This dissertation focuses on increasing the reliability and versatility of the MCO/MS method as a tool for studying metal-protein interactions. The importance of ascorbate concentration in controlling the oxidation yield and specificity of the MCO reactions has been established, and sodium persulfate has been identified as a specific and potent oxidant. Greater insight into the role of ascorbate has allowed us to develop a new "detuned" version of MCO/MS method that can oxidize residues beyond the metal-binding site, making the method more sensitive to minor changes in protein structure that might occur upon metal binding to a protein. Application of the detuned method to Cu binding of β-2-microglobulin suggests some possible structural changes caused by metal binding that might provide insight into the amyloid formation of this protein. The versatility of the MCO/MS approach has been expanded by, improving the speed of the method and extending its application to other metals. In an effort to better study dynamic systems, microwave irradiation has been used to decrease the time required for MCO reactions by a factor of 10. The utility of the MCO/MS method for the study of non-Cu transition metal systems has been illustrated by determining suitable MCO reaction conditions for peptides that bind Mn, Fe, Co and Ni and a whole protein, Nickel superoxide dismutase. Finally, we have studied the effect of amino acid oxidation on the dissociation patterns of peptide ions during tandem MS (MS/MS) experiments and the identification of binding residues. We have found that histidine oxidation changes the peptides dissociation pattern. We demonstrate that an N-terminal derivatization method can be used to simplify the MS/MS interpretation in such circumstances.
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"Insights into the Role of the Metal-Binding Sites of Quercetin 2,3-Dioxygenase from Bacillus subtilis." Doctoral diss., 2010. http://hdl.handle.net/2286/R.I.8650.
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abstract: The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future.Dissertation/Thesis
Ph.D. Biochemistry 2010
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Geeganage, Sandaruwan. "Mechanistic analysis of variant galactose-1-phosphate uridylyltransferases incorporating amino acid-mutations in the active and metal binding sites." 1999. http://catalog.hathitrust.org/api/volumes/oclc/43303261.html.
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Sui, Meng-Jiun, and 隋孟君. "Characterization of the divalent metal ion and the phosphate ion binding sites in the H-N-H endonulease colicin E7." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/56562691591829371051.
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博士國防醫學院
生命科學研究所
91
Colicins are a group of plasmid-encoded protein toxins produced by Escherichia coli to kill other sensitive E. coli and closely related coliform bacteria. The cytotoxic activities of colicins against their target cells are well documented: as an RNase, a DNase, a protein synthesis inhibitor or an ionophore. The subfamily of E-group DNase-type colicins, including colicin E2, E7, E8 and E9, contain an H-N-H motif in the cytotoxic nuclease domain, capable of cleaving DNA nonspecifically. This H-N-H motif was firstly identified in a subfamily of homing endonucleases, which cleave DNA site specifically and initiate a process of transferring a mobile intervening sequence into a homologous allele that lacks the sequence. The H-N-H motif contains two anti-parallel b-strands linked to a C-terminal a-helix, with a divalent metal ion located in the center. It is however not clear how the H-N-H motif mediates its function in DNA binding and hydrolysis. For a better understanding of the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 in complex with its inhibitor Im7 (nuclease-ColE7/Im7) in two different crystal forms and we resolved the structures of EDTA-treated, Zn2+-bound and Mn2+-bound complexes in the presence of phosphate ions at resolutions of 2.6 to 2.0. Å. The structure study of the EDTA-treated nuclease-ColE7/Im7 offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. We showed that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg2+-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in H-N-H motif. However a Zn2+ or Mn2+ ions were observed in the center of the H-N-H motif in cases of Zn2+ or Mn2+-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis. We also construct a structural model for the nuclease-ColE7 bound to DNA and propose a mechanism for DNA hydrolysis catatalyzed by the H-N-H endonucleases.
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Bilder, Patrick Wallace. "The structural diversity of metal binding sites in bacterial metalloproteins : the disordered iron-binding coil of iron-sulfur cluster protein A and the stable zinc ribbon motif of the carboxyltransferase subunit of acetyl-coa carboxylase." Diss., 2005. http://etd.library.vanderbilt.edu/ETD-db/available/etd-01222006-213113/.
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Dias, Alistair Vincent Michael. "The control and utilization of Ni(II) in Escherichia coli: The Ni(II)-specific response of NikR and the characterization of the metal-binding sites of HypB." 2009. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=968401&T=F.
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Azizan, Nur. "Study of metal binding site of methyl parathion hydrolase." Phd thesis, 2015. http://hdl.handle.net/1885/149587.
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Metal ions are an essential component of numerous proteins. In some cases they have a structural role, but their more important roles are redox proteins and enzymes. Metals interact quite differently with proteins depending upon the function of the protein. In enzymes metals are coordinated to the protein, but maintain vacant coordination sites to allow them to interact with substrates or to activate nucleophiles for attack. In binuclear metalloenzymes, metals have the potential to function in both capacities where they can bind substrate and can also activate water for attack. Most studies of binuclear metalloenzymes focus on the native enzyme how it might interact with the substrate. Clearly, the interaction between protein and substrate depends critically upon the metal centers. This study sets out to study the metal centers, to identify the role of particular residues in binding metals and the effect of perturbing the metal centers on catalytic activity. This study was never intended to make a detailed study of catalytic activity, but rather to provide background information for such studies. It was also hoped that data collected during the course of this study might shed some light on previous mechanistic studies. To achieve the objected outlined above a suitable enzyme was selected for study, which is methyl-parathion hydrolase (MPH). Its active site residues were mutated and the resulting protein variants were subjected to a variety of experiments. The effects of mutations on metal binding affinity, catalytic activity as well as overall protein stability were determined. Very early in the study, it became clear that metals ions had a significant effect on protein stability and that the apo-protein was much less stable that the fully metal bound form. Some mutations had a profound effect on activity yet caused minor changes in stability while other mutants exhibited the opposite effect. Surprisingly, in some cases removing the side-chain of a coordinating group had little effect on the affinity of the protein for the metal or on its catalytic activity. However, it was also clear that two metal ions were essential for activity; little activity was observed with a single metal ion bound to the protein. MPH has evolved to accommodate two metal ions in its active site such that minor alterations to the coordinating residues results in a loss of either stability or activity. These observation suggest that MPH has evolved to optimize activity as well as stability; the latter being important for the long-term ability of the enzyme to be an efficient catalyst.
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Wei, Chieh-Hwa, and 韋建華. "Identification of the Metal Binding Site of Pigeon Liver Malic." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/45443903656679936060.
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Lin, Jau-Ji, and 林肇基. "Prediction of Metal-Binding Site Residues Using Support Vector Machine." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/82371337700374814949.
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碩士國立交通大學
生物資訊研究所
93
Correct identification and analysis of the metal-binding site provides useful clues to the modeling and designing of the binding site in proteins for industrial and therapeutic purposes. As the number of the biological data is rapidly accumulated, the use of machine learning approach to do the prediction becomes more reliable now than ever. We have developed a method using support vector machine (SVM) to predict the metal-binding site residues in proteins containing metal ions. The information used to encode the site residues includes sequence profiles and structural features. The results show that the use of buffer zone can effectively improve the true positive rate (TPR) of the prediction. On five-fold cross-validation, we obtain an average prediction accuracy of 97.4% and 46.2% TPR at a 5% false positive rate (FPR). The results indicate that the use of SVM with suitable coding schemes is an effective way to predict the metal-binding sites in proteins.
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Shou-YunLu and 盧守筠. "Site-Directed Mutagenesis Studies on the Divalent Metal Ions Binding Site of Rat Lens αB-Crystallin." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/fwjydk.
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博士國立成功大學
化學系
102
The lens αB-crystallin, playing a major role in maintaining the transparency of the eye lens, possesses chaperone-like function to prevent the lens proteins from aggregation due to foreign stress and/or aging. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. The role of some metal ions in the αB-crystallin biology has been reported. Theoretical calculations have proposed that the coordination site of human αB-crystallin for binding divalent metal ions were His101, His119, Lys121, His18 and Glu99. In this study, H18G, H119G, and H101G mutant types of rat lens αB-crystalin were cloned and expressed to investigate whether His18, His119 and His101 are the coordination binding sites. And the results suggested that His18, His119 and His101 were the crucial binding sites for Cu (II) and Zn (II) in terms of chaperone-like activity and structure. Copper and zinc at 1 mM concentration significantly increase the chaperone-like activity in wild type αB-crystalin, whereas zinc, copper and magnesium at 1 mM reduced the activity of mutant type significantly. ANS fluorescence measurement showed that there was no linear relationship between chaperone-like activity and surface hydrophobicity, indicating that surface hydrophobicity is not prerequisite for exhibiting chaperone-like activity. Both the Far- and Near-UV CD spectra suggested that the wild type reflected more β-sheet structural characteristics; whereas the mutant type reflected more random coil characteristics and more micro-environmental changes around the tryptophan residues. The results from chaperone-like activity, ANS fluorescence measurement and Far- and Near-UV CD studies indicate that the replacement of His18, His119 and His101 with Glycine resulted in a conformational and minor environmental changes that decrease chaperone-like activity in the presence of divalent ions suggested that His18, His119 and His101 were a crucial binding site for Cu (II) and Zn (II), respectively. All results together suggest that His18, His119 and His101 coordinate each other for the binding site of Cu (II) and Zn (II) in terms of improving the chaperone-like activity and stability of crystallin/metal ion complex, which further confirmed the proposed binding site based on the theoretical calculation.
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Gavrilova, Anna Leonidovna. "One-site addition two-metal oxidation reactions of unsymmetrical bimetallic complexes related to dioxygen binding by hemerythrin /." 2003. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3108077.
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Chen, Wei-Nan, and 陳威男. "Evalution of Glutamyl 234 Residue in the Metal Binding Site of Pigeon Liver Malic Enzyme." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/94295390193799499427.
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碩士國防醫學院
生物化學研究所
89
Pigeon liver malic enzyme (EC 1.1.1.40) catalyzed the oxidative decarboxylation of L-malate to CO2 and pyruvate, with concomitant reduction of NDAP+ to NADPH. Glu234 is one of highly conserved amino acid residues among several species. Site-directed mutagenesis method was use to confirm the role of Glu234 in pigeon liver malic enzyme. The preliminary kinetic studies revealed that the apparent KmMn and KmMalate value for E234A were increased by about 300-fold and 15-fold, respectively. From the initial velocity experiment, the KmMn and KdMn of E234A were increased to 450-fold and 620-fold of that of wild type. We proposed that E234A was one of the divalent metal chelate sites of the malic enzyme. In the presences of saturated substrates and cofactor, E234A demonstrated a downward curve instead of a straight line observed in wild type in the kinetic activity assay. The Ki values of NADPH, pyruvate and sodium bicarbonate for E234A were 8 mM, 49 mM and 78 mM, respectively. These values were similar to those for wild type. E234A was more stable than wild type at 30°C. Therefore, the downward curve was not resulted from product inhibition and enzyme stability. Acrylamide quenching studies showed that E234A did not demonstrate a slow conformational change in the presence of Mn2+. The formation of NADP-pyruvate nonproductive adduct was proposed to explain the phenomenon of the downward curve.