Tesis: "Metal-binding proteins"

1

Flowers, Andrew E. "Metal-binding proteins in tropical marine invertebrates". Thesis, Queensland University of Technology, 1995.

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2

Hennig, Helmke Friedrich-Karl Otto. "Baseline surveys and metal binding proteins as metal pollution indicators". Doctoral thesis, University of Cape Town, 1985. http://hdl.handle.net/11427/22479.

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Bibliography: pages 304-309.
The field of metal determination as a part of pollution studies, has been critically examined and metal pollution may be defined in one simple statement: The presence of metal binding proteins confirms toxic metal pollution. It has been shown that current methods of metal determination in biological systems are of little use. This has been illustrated by both a review of metal concentration in Southern African coastal water, sediments and biotopes, and by a comparative baseline study of organisms from Gough Island and Mar ion Island. These showed that extrapolation of results from one geographical area to another are invalid and that this interpretation is made difficult by factors such as age, sex, size life stage of the organisms. Furthermore, it was shown that many reports on metal pollution do not even mention fundamental information such as the size or the sex of the animals. Metal pollution could be linked to metal binding protein through an independent pollution er i ter ia, for example, the out of season moulting of crayfish. The new definition of metal pollution has then been tested by application to five different organisms (crayfish, Jasus lalandii; hermit crab, Diogenes brevirostris; shrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis and limpet, Patella granularis) kept under identical conditions and it was shown that a much more meaningful interpretation of the results could be made. The new definition was al so tested with two naturally occurring metal accumulating organisms (whelk, Bullia digitalis and "kikuyu" grass) and it was shown that dramatic increases in metal may not necessarily be toxic. It was concluded that less effort and time should be spent on metal analysis in determination of metal pollution and attention should rather be directed to the presence or absence of metal-binding proteins.

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3

Limson, Janice Leigh. "Electrochemical studies of metal-ligand interactions and of metal binding proteins". Thesis, Rhodes University, 1999. http://hdl.handle.net/10962/d1018239.

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Electrochemical methods were researched for the analysis of metals, proteins and the identification of metal binding proteins. Adsorptive cathodic stripping voltamrnetry for metal analysis combines the inherent sensitivity of electrochemical techniques with the specificity of ligands for the nonfaradaic preconcentration of analytes at the electrode. The utility of catechol, resorcinol, 4-methylcatechol and 4-t-butylcatechol as ligands was explored for the sensitive analysis of copper, bismuth, cadmium and lead on a mercury film glassy carbon electrode. Metal complexes of lead, copper and bismuth with resorcinol showed the largest increase in current with increase in metal concentration, whereas complexes of these metals with 4-t-butylcatechol showed the lowest current response. Cadmium showed the highest current responses with 4-methylcatechol. The four metals could be determined simultaneously in the presence of resorcinol, although considerable interference was observed between bismuth and copper. The electroanalysis of cysteine and cysteine containing proteins at carbon electrodes are impaired by slow electron transfer rates at carbon electrodes, exhibiting high overpotentials, greater than 1 V vs Ag! Agel. Metallophthalocyanines have been shown to promote the electrocatalysis of cysteine at lowered potentials. Chemical modification of electrodes with appropriate modifiers is a means of incorporating specificity into electroanalysis, with applications in electrocatalysis. A glassy carbon electrode was modified by electrodeposition of cobalt (II) tetrasulphophthalocyanine [Co(II)TSPct to produce a chemically modified glassy carbon electrode (CMGCE). The CoTSPc-CMGCE catalysed the oxidation of cysteine in the pH range 1 to 10. The significance of this electrode is an application for analysis of proteins at biological pH's. A biscyanoruthenium(II) phthalocyanine CMGCE catalysed the oxidation of cysteine at 0.43 V vs Ag/AgCl a significant lowering in the overpotential for the oxidation of cysteine. Metallothionein, a metal binding protein, is believed to be involved in metal homeostasis and detoxification in the peripheral organs of living systems. A method for the quantitative determination of this protein utilising its high cysteine content was presented. At pH 8.4 Tris-HCl buffer, and using a CoTSPc-CMGCE modified by electrodeposition of the modifier, the anodic peaks for the oxidation of metallothionein was observed at 0. 90 V vs Ag/ AgCI. Ferredoxin is a simple iron-sulphur protein. One tenth of its residues are cysteine. Ferredoxin is involved in simple electron transfer processes during photosynthesis and respiration. Electrochemical studies of spinach ferredoxin were conducted at a CoTSPc-CMGCE. Anodic currents for the oxidation of the cysteine fragment of ferredoxin was observed at 0.85 V vs Ag/AgCl in HEPES buffer at pH 7.4, representing a new method for analysis of this protein. Voltammetric studies of its ferric/ferrous transition have shown quasi-reversible waves atE~ -0.62 V vs Ag/AgCl only in the presence of promoters. At a CoTSPc-CMGCE, a cathodic wave attributed to the reduction of Fe(III)/Fe(II) was observed at Epc -0.34 V vs Ag/AgCl. This represents an alternative method for voltammetric studies of the ferric/ferrous transition at significantly lowered potentials. Melatonin, a pineal gland hormone functions m setting and entraining circadian rhythms and in neuroprotection as a free radical scavenger and general antioxidant. Using adsorptive cathodic stripping voltammetry, the binding affinities of melatonin, serotonin and tryptophan for metals, were measured. The results showed that the following metal complexes were formed: aluminium with melatonin, serotonin and tryptophan; cadmium with melatonin and tryptophan; copper with melatonin and serotonin; iron (III) with melatonin and serotonin; lead with melatonin, tryptophan and serotonin, zinc with melatonin and tryptophan and iron (II) with tryptophan. The studies suggest a further role for melatonin in the reduction of free radical generation and in metal detoxification and may explain the accumulation of aluminium in Alzheimer's disease.

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4

Wheeler, Lucas. "The Evolution of Metal and Peptide Binding in the S100 Protein Family". Thesis, University of Oregon, 2018. http://hdl.handle.net/1794/23178.

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Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.

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5

Lindsay, William Pirie. "Expression of recombinant metal-binding proteins in E. Coli and in Synechococcus PCC7942 : examination of metal binding in vivo and in vitro". Thesis, Durham University, 1992. http://etheses.dur.ac.uk/5727/.

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Metallothioneins (MTs), cysteine-rich proteins and polypeptides, are proposed to detoxify excess intracellular metal ions via sequestration. Three genes, each encoding a protein related to this group of molecules, were expressed in Escherichia coli and in Synechococcus PCC7942 (variant PIM8) in order to examine the metal binding properties of their products. Phenotypic alterations, in terms of metal-tolerance and - accumulation, were assessed in cells expressing these genes. The genes which were expressed were: (1) smtA from Synechococcus PCC7942, which is designated to be the first isolated prokaryotic MT gene; (2) PsMT(_A), a gene from pea (Pisum sativum L) which encodes a protein with similarity to class I MT; and (3) a synthetic gene encoding(Glu-Cys)(_3)Gly, an analogue of the phytochelatin (PC; class III MT) molecule (γGlu-Cys)(_3)Gly. The protein encoded by smtA was shown to have high affinity for metal ions (Hg, Cd, Cu, Zn), supporting the designation of smtA as a prokaryotic MT gene. Comparison with mammalian MT revealed that the affinity of the product of smtA for Zn was higher than that of the mammalian protein, suggesting a role for this protein in Zn homoeostasis and/or detoxification in Synechococcus sp. E. coli cells expressing smtA exhibited increased accumulation of Zn and Cd (3-fold and 1.4-fold respectively relative to control cells), but no increase in tolerance toward Zn, Cd or Cu. Comparison of the metal-affinity of the product of PsMT(_A) with that of mammalian MT revealed that this protein also has high affinities for Cu, Cd and Zn. These data support the hypothesis that PsMTp^ is a higher plant MT gene. Affinity of the product of this gene for Cu was higher than that of mammalian MT, suggesting a role for this protein in Cu homoeostasis and/or detoxification. Expression of PsMT(_A) in E. coli resulted in increased accumulation of Cu, Cd and Zn. Cu accumulation was increased more substantially than either Zn or Cd accumulation in cells expressing PsMT(_A). No increase in tolerance toward any of these metals was observed in E. coli expressing this gene. There is evidence that PCs are involved in Cd detoxification in higher plants. Genes encoding enzymes involved in the synthesis of these molecules have not been isolated, precluding gene transfer experiments for investigation of their function. Expression of a gene encoding (Glu-Cys) gGly in E. coli resulted in increased tolerance toward Cd, but not Cu or Zn. Thus, a predicted function of a secondary metabolite (PC) was observed when a gene product based on the structure of this molecule was expressed in a heterologous system. No significant increase in accumulation of Cd, Cu or Zn was detected in cells expressing this gene.smtA transcripts were shown to increase in abundance in response to elevated concentrations of Cd in Synechococcus PCC7942 (variant PIM8). Sequences derived from the smt locus were fused to a synthetic gene encoding (Glu-Cys)(_3)Gly, and introduced into Synechococcus PCC7942(variant PIM8). Transcripts encoding (Glu-Cys)gGly increased in response to exposure of these cells to Cd. Cells containing this construct exhibited increased tolerance toward Cd. Data concerning expression of(Glu-Cys)(_3)Gly in E. coli and Synechococcal cells support the hypothesis that PCs may have a role in detoxification of excess intracellular Cd. Comparison of the data obtained in these studies has been used to assess the factors affecting metal-accumulation and -tolerance as a result of expression of heterologous metal-ligands in microbial cells.

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6

Wang, Xiaoyan. "Synucleins and their roles in the pathology of Parkinson's disease as metal binding proteins". Thesis, University of Bath, 2009. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512329.

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α-synuclein is an abundant and conserved presynaptic brain protein (Uversky 2007). It has received extensive attention since its aggregation was identified as the main component of Lewy bodies and Lewy neurites, which is the pathological hallmark of several neurodegenerative diseases, collectively known as synucleinopathies, including Parkinson's Disease (PD) (Uversky 2007). Considerable information has been collected about the structural properties and conformational behavior of α-synuclein, although the precise function is still under investigation. Metal ions such as copper and iron, can accelerate the aggregation and fibrillation of α-synuclein. Metal ions may exert their dual physiopathological properties through the interaction with α-synuclein, converting protein structure and/or inducing oxidative stress. In this study, isothermal titration calorimetry and electron paramagnetic resonance were used to determine the metal-binding property of the synuclein proteins, proving the presence of four Cu(II) binding sites per molecular of α-synuclein, with the coordination modes of 1N3O and 2N2O. Furthermore, α-synuclein has a catalytic action on the redox cycling of Cu(II), which was assessed by the application of cyclic voltammetry. However, this property is absent on β-synuclein and γ-synuclein, which belong to the synuclein family and have been suggested to be the physiological regulators of α-synuclein expression. In vivo, immunofluoresence studies revealed that Cu(II) increases the aggregates formation in mammalian doperminergic neuron cells overexpressing α-synuclein and the PD-associated mutants, while no aggregates have been found in cells overexpressing β-synuclein and γ-synuclein.

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7

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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8

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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9

Fallas, Andrea Jennifer. "C1, SAP and ZiCo : structural studies of three metal‑binding proteins from a crystallographic perspective". Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/7414/.

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Atomic resolution models of proteins are crucial for understanding their biological mechanisms and provide insights into the relationship between protein sequence and structure. Many protein structures incorporate metal ions, exploiting their unique chemistry as reaction centres or for structural stability. This thesis describes the progress made towards solving the structures of three such metal-binding proteins by means of X-ray crystallography. Complement component C1 is a large protein complex that initiates the first line of immune defence and requires calcium for structural stability. Fragments of C1 have already been solved at high resolution, but there are no accurate models of the assembled complex. In this work, a new method for purifying intact C1 from human serum was developed and the purified complex was characterised by various methods. Finally, attempts were made to crystallise native human C1 with a view to obtaining high-resolution structures of the entire complex. Serum amyloid P is another serum protein, also thought to be involved in the immune response. It is often found associated with amyloid deposits, although SAP binds a variety of ligands in a calcium-dependent manner. While the structure of SAP has been determined, its physiological function is still not fully understood. SAP was purified using established methods and its ligand-binding properties were investigated under various conditions using dynamic light scattering, in an attempt to gather more information about the possible function of this molecule. Finally, ZiCo is a small peptide that was designed to switch between a multimeric coiled coil and a monomeric zinc finger fold on binding zinc. The system has been characterised extensively in solution, but high-resolution structures are required to validate the design. ZiCo was crystallised and diffraction data were collected. The structure of the peptide was partially solved, indicating that the multimeric form of the ZiCo peptide is indeed a trimeric coiled coil.

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10

Roy, Poorna Roy. "Analyzing and classifying bimolecular interactions:I. Effects of metal binding on an iron-sulfur cluster scaffold proteinII. Automatic annotation of RNA-protein interactions for NDB". Bowling Green State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1496412736120654.

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11

Calatayud, Robert Sara. "Modular evolution of domain repeat proteins. Metal-binding and domain repeats of Metallothioneins in molluscs and chordates". Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/673942.

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Proteins are composed of domains, a well-defined region within a protein that constitutes a stable, independently folding, compact structural unit, and that usually performs a specific function. During protein evolution, domains have been used as a ‘modules’, and the vast majority of the current proteins show a modular organization in which two or more domains are combined. While the creation of multi-domain proteins through shuffling different domains has been studied extensively, the evolution of proteins made of tandem repeats of a same domain has been less investigated. It is known, for instance, that the fraction of proteins with domain repeats is higher in multicellular organisms than in unicellular organisms, or that proteins with domain repeats seem to be related with stress response functions or with the acquisition of high yielding capacity, but the origin, the genetic mechanisms and the evolutionary forces controlling the expansion of domains repeats in modular proteins are still poorly understood. To investigate the functional and structural evolution of modular proteins with domain repeats, we have chosen the Metallothioneins (MTs) as a case study. MTs are present across all the tree of life and due to their metal-binding ability have been involved in metal homeostasis and detoxification of many organisms. They are modular cysteine-rich proteins that bind metal ions through functionally and structurally independent domains that form metal-thiolate clusters. Many MTs are bi-modular proteins made of a tandem repetition of two similar (although not identical) metal-binding domains. There also are large multi-modular MTs that have expanded the number of their domain repeats, and therefore, their metal-binding capacity. Studying the evolution of metallothionein domains in several phyla can help us to understand, at some level, the evolvability of organisms to adapt to new environmental conditions. In our studies we have identified, analysed, characterized and compared many MTs from different groups of selected organisms (chordates and molluscs) in order to study their domain composition and arrangement, and to untangle the evolution of their modular organization.
Las proteínas están compuestas por dominios, una región bien definida dentro de una proteína que constituye una unidad estructural compacta, estable y que se pliega independientemente, y que normalmente realiza una función específica. Durante la evolución de las proteínas, los dominios se han utilizado como "módulos", y la gran mayoría de las proteínas actuales muestran una organización modular en la que se combinan dos o más dominios. Si bien la creación de proteínas de múltiples dominios mediante la combinación aleatoria de diferentes dominios se ha estudiado ampliamente, la evolución de proteínas compuestas de repeticiones en tándem de un mismo dominio se ha investigado menos. Se sabe, por ejemplo, que la fracción de proteínas con dominios repetidos es mayor en organismos multicelulares que en organismos unicelulares, o que las proteínas con dominios repetidos parecen estar relacionadas con funciones de respuesta al estrés o con la adquisición de una alta capacidad productiva, pero el origen, los mecanismos genéticos y las fuerzas evolutivas que controlan la expansión de las repeticiones de dominios en las proteínas modulares aún son poco conocidos. Para investigar la evolución funcional y estructural de las proteínas modulares con dominio repetido, hemos elegido las metalotioneínas (MT) como caso de estudio. Los MT están presentes en todo el árbol de la vida y, debido a su capacidad de unión a metales, han estado involucrados en la homeostasis de metales y la desintoxicación de muchos organismos. Son proteínas modulares ricas en cisteína que se unen a iones metálicos a través de dominios funcional y estructuralmente independientes que forman grupos de tiolatos metálicos. Muchos MT son proteínas bimodulares hechas de una repetición en tándem de dos dominios de unión a metales similares (aunque no idénticos). También hay grandes MT multimodulares que han ampliado el número de repeticiones de su dominio y, por lo tanto, su capacidad de unión a metales. El estudio de la evolución de los dominios de las metalotioneínas en varios filos puede ayudarnos a comprender, hasta cierto punto, la capacidad de evolución de los organismos para adaptarse a nuevas condiciones ambientales. En nuestros estudios hemos identificado, analizado, caracterizado y comparado muchos MT de diferentes grupos de organismos seleccionados (cordados y moluscos) para estudiar la composición y disposición de sus dominios y desentrañar la evolución de su organización modular.

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Pedersen, Sindre Andre. "Metal binding proteins and antifreeze proteins in the beetle Tenebrio molitor : a study on possible competition for the semi-essential amino acid cysteine". Doctoral thesis, Norwegian University of Science and Technology, Department of Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-1504.

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In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are involved in these defence systems (Korsloot et al. 2004). Since an organism’s resources may be considered to be limited, the ability to restore homeostasis depends on the severity of the different forms of stress it experiences. It has been proposed that pollutants present in the environment may alter the ability to respond to climatic stressors like e.g. low temperature, desiccation (Holmstrup 2002).

This work deals with the possible consequences of combined stress from metal exposure and low temperature in cold hardy insects. Many of these insects produce so called antifreeze proteins that protect them from lethal freezing. Metallothioneins are metal binding proteins that are considered to be important in detoxification when animals are exposed to metals. Metallothioneins and most forms of antifreeze proteins from insects are known to contain unusually high amounts cysteine. Cysteine is considered to be semi-essential, since it must be derived from the essential amino acid methionine (Choen 2004). Induction of one of these two types of proteins may potentially deplete the cysteine pool and thus reduce the capacity to produce the other type. Alternatively, the animals might have evolved other structures to avoid a potential competition for cysteine. The purpose of the present work was to explore these possible scenarios.

Paper I and II reprinted with kind permission of Elsevier, Sciencedirect.com

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13

Tsang, Cheuk-nam y 曾卓南. "Mining of proteins and motifs associated with bismuth binding and monitoring metal uptake in helicobacter pylori by metallomics". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46503535.

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14

Howard, Warren A. "Synthesis and characterisation of platinum(II) and ruthenium(II) polyamide conjugates". Thesis, View thesis, 2008. http://handle.uws.edu.au:8081/1959.7/43899.

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This thesis reports the synthesis and characterisation of two novel metallo-polyamide conjugates; LLSP4-Pt and LLSP4-Ru. Several synthetic strategies were employed in the development of the polyamides, beginning with solution phase chemistry. Construction of the polyamide was attempted via chain elongation with sequential additions of pyrrole monomers, however, solution phase methods were unable to achieve the coupling of four consecutive pyrrole rings. Solid phase chemistry, using a peptide synthesiser, was then used as an alternative. Synthesis using solid phase chemistry required the production of the monomeric building block Fmoc-py-COOH using adaptations of various literature methods and was successfully characterised by 1H NMR. The proof-of-concept molecule LLSP4-DPA was the first polyamide made using Fmoc-β-Alanine-OH-WANG resin although several impurities from the cleaving agent 3-dimethylaminopropylamine persisted despite attempts at purification. The solid phase support was then changed to chlorotrityl resin and was used to synthesise the precursor polyamide LLSP4 with high purity. LLSP4-Pt was then successfully made using chlorotrityl resin and characterised by NMR (1H and 195Pt), ESI-MS and elemental analysis. The ligands dpq, 4-CO2H-phen and intermediates thereof, were successfully synthesised and used in the production of the ruthenium conjugate. The precusor LLSP4-(4-CO2H-phen) was made using the same solid phase techniques employed for LLSP4-Pt. Coordination of [Ru(dpq)2Cl2] to LLSP4-(4-CO2H-phen) was afforded by heating in EtOH producing LLSP4-Ru which was characterised by 1H NMR and ESI-MS. Preliminary studies showed that upon addition of DNA to LLSP4-Ru a large increase in fluorescence is seen which suggests an intercalative binding mode. DNA binding studies for LLSP4-Pt were conducted using CD based titrations with ct-DNA. The binding constant of LLSP4-Pt was found to be 4.1 × 105 M-1 with a binding site size of 4 base pairs. The ability LLSP4-Pt to form coordinate covalent bonds with guanosine nucleosides was investigated and monitored by 1H NMR. Incubation of guanosine with LLSP4-Pt shows a new resonance for H1' is observed at 5.98 ppm while two new resonances for H8 are observed at 8.78 and 8.79 ppm.

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15

Howard, Warren A. "Synthesis and characterisation of platinum(II) and ruthenium(II) polyamide conjugates". View thesis, 2008. http://handle.uws.edu.au:8081/1959.7/43899.

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Thesis (Ph.D.)--University of Western Sydney, 2008.
A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

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De, Angelis Fabien. "Characterization of proteins involved in RND-driven heavy metal resistance systems of Cupriavidus metallidurans CH34". Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210154.

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Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ;cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it.
Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished

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Phillips, Christine M. (Christine Marie). "Relating metal binding to DNA binding in the nickel regulatory protein NikR". Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57569.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Vita. Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The concentration of transition metals within the cell must be tightly regulated. If the concentration of a given transition metal is too low the cell may not be able to perform life-sustaining processes, while high levels of metals are poisonous to the cell and can cause cell death. In Escherichia coli, NikR regulates nickel uptake by blocking transcription of the genes encoding the nickel uptake transporter, NikABCDE. NikR is a homotetrameric transcription factor with a central metal binding domain (MBD) that includes the tetrameric interface and two flanking dimeric ribbon-helix-helix (RHH) DNA-binding domains. Early work revealed that NikR can bind a variety of transition metal ions and has two binding affinities for the nik operon: nM when stoichiometric Ni2+ binds NikR and pM when excess Ni2+ binds. The enhanced DNA affinity suggests the presence of low affinity nickel binding sites on the protein. Recently, it has been shown that NikR also requires K+ to bind DNA, suggesting yet another type of metal binding site on the protein. To understand NikR's ability to bind multiple transition metal ions and how Ni2+ specifically induces NikR-DNA binding, we solved the crystal structures of the apo- MBD and BMD bound to Zn2+ and Cu2+. Comparing these structures to the previously published Ni2+-MBD structure, we noted that when the proper metal binds to NikR it utilizes H76 of alpha helix 3 as a ligand. This, in turn, orders helix !3, and we propose this conformational stabilization is a key step in the NikR-DNA binding mechanism. Electrostatic free energy calculations and thermodynamic integration were used to study which metal prefers to bind at a site between the MBD and RHH domains that is formed when NikR is bound to DNA. Our studies illustrate that NikR-DNA binding was most favorable when this site contains a monovalent cation the size of K+. These studies support a physiological role of K+ in NikR-DNA binding. Structures from crystals of NikR and NikR-bound to DNA soaked with excess nickel ions indicate six types of potential low-affinity nickel binding sites on the protein surface. Binding of excess nickel ions to these sites does not induce any significant conformational change, suggesting that these sites have an electrostatic effect increasing ! 4 NikR's affinity for DNA. Using a combination of X-ray crystallography and molecular simulations we have identified and explored the metal binding sites on E. coli NikR and how they influence NikR:DNA binding.
by Christine M Phillips.
Ph.D.

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18

Klewpatinond, Mark. "Spectroscopic investigation of metal binding to the prion protein". Thesis, Queen Mary, University of London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.500024.

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19

Shahir, Shafinaz. "Engineering and the maltose binding protein for metal ions sensing". Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434921.

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20

Krauss, Oliver. "Structural studies on glycerol dehydrogenase from Bacillus stearothermophilus". Thesis, University of Southampton, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243048.

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21

Han, Ji Hoon. "Fluorescent Nucleobases for Studying DNA Structure, Protein Interaction and Metal Binding". Kyoto University, 2019. http://hdl.handle.net/2433/242637.

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22

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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23

Green, Andrew F. D. "Metal ligation in ZIF268, a zinc finger protein, effects on DNA binding". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ45850.pdf.

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24

Stevens, Daniel J. "Metal binding behavior of the prion protein and relevance to disease progression /". Diss., Digital Dissertations Database. Restricted to UC campuses, 2008. http://uclibs.org/PID/11984.

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25

Allen, Mark Andrew. "Protein Cage Architectures as a Nano-Platform for Material Synthesis and Metal Binding". Thesis, Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/allen/AllenM0806.pdf.

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Supramolecular proteins that assemble into cage like architectures have been used for nano-material synthesis and as a scaffold for metal binding. Material synthesis can be performed by exploiting the cage-like properties of these nano-containers and relying on the electrostatically distinct interior environment that drive mineral encapsulation. Ferritin and ferritin like proteins can be used as size constrained reaction vessels that encapsulate materials that have sizes that are determined by the internal dimensions of the protein cage. These range from 5 nm for the ferritin like protein from Listeria innocua to 24 nm for the interior of an engineered plant virus (Cowpea chlorotic mottle virus). Inorganic materials synthesized within these constrained reaction volumes are monodisperse in size. The crystallinity and phase of material prepared is determined by the reaction conditions, which are mild compared to other preparative methods. The metal binding affinity of certain viral protein cages allows the study of the role that metals play in such processes as viral assembly and infection as well as transmission. If paramagnetic metal ions are bound to the viral protein cage, the biological scaffold has potential use as an MRI contrast agent. Here multiple protein cage platforms are discussed with an emphasis on engineering non-native functionality to many of the protein cages. Engineering nonnative function to protein cages involves both genetic and chemical modification for the purpose of increasing stability and changing electrostatic interactions. Together these modifications serve to reinforce the versatility of protein cage architectures for both mineral synthesis and metal binding.

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26

Nguyen, Hieu H. "Water Remediation by Metal Binding Protein| A Method to Offset Environmentally Hazardous Practices". Thesis, California State University, Long Beach, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10784348.

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The acquisition of fossil fuels has many harmful effects on the environment. One of those effects is the release of toxic heavy metals, which may find its way to water reservoirs or ecosystems. Metallothionein (MT) is a metal binding protein that has potential to alleviate such metal pollutions by scavenging for these metals. It can also be used to collect scarce metals used in industrial practices. The results showed that MT is able to bind metals in an organic environment, and that MT may be induced to release metals with the addition of acid. It was also found that the amount of metal bound is proportional to the amount of MT used. Despite efforts to show that MT may be used in PUREX, results indicate that the protein is unlikely to be used for this purpose.

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27

Chaton, Catherine T. "Metal Binding Specificity and N-terminal Function of the Staphylococcal Biofilm Protein Aap". University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin151092604272917.

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28

Gardner, Stewart G. "Studies of PhoU in Escherichia coli: Metal Binding, Dimerization,Protein/Protein Interactions, and a Signaling Complex Model". BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/5685.

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Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate and that phosphate signal is passed through PhoU to PhoR. Further, this work characterizes residues important for interaction on PhoU and PhoR and identifies a structural model for interaction. This model points to a potential mechanism for PhoU mediated signaling to PhoR. We tested this model with direct coupling analysis and obtained further confirmation. Further use of these techniques may elucidate more of the interactions necessary for proper phosphate signaling.

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29

Kirberger, Michael Patrick. "Analyses and Applications of Metalloprotein Complexes". Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/chemistry_theses/14.

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The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.

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30

Loftin, Isabell. "Structural and Biochemical Studies of the Metal Binding Protein CusF and its Role in Escherichia coli Copper Homeostasis". Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/193875.

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Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.

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31

Grady, Amanda Ellen. "The regulation of the type 5 cyclic nucleotide phosphodiesterase in airway smooth muscle by metal ions and small molecular weight proteins". Thesis, University of Strathclyde, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366810.

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32

King, Jennifer Ellen. "Expression, purification and metal-binding properties of Î±-synuclein, a protein implicated in Parkinson's disease". Thesis, Lancaster University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435870.

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33

Abreu, Neto João Braga de. "Identificação e caracterização do gene OsPMBP1 (Prenylated Metal Binding Protein 1) de arroz (Oryza sativa)". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2009. http://hdl.handle.net/10183/24070.

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O número de genes atualmente identificados no genoma do arroz (Oryza sativa ssp japonica) é de cerca de 32.000. Graças aos esforços de diferentes grupos ao redor do globo, o papel de muitos desses genes é melhor compreendido no momento. A função de um gene pode ser inferida principalmente pela análise de seu padrão de expressão, da estrutura da proteína codificada e pela análise de mutantes nos quais o gene se encontre interrompido, silenciado ou super-expresso. Para esse propósito, nosso grupo de pesquisa vem desenvolvendo linhagens transgênicas pela inserção de uma construção de T-DNA baseada no sistema de transposons de dois componentes iAc/Ds. Uma dessas linhagens (Ds72) apresenta estatura reduzida e sua inserção de T-DNA interrompe um locus, cuja função gênica ainda é desconhecida. O objetivo desse trabalho é a identificação e caracterização desse gene, que denominamos de OsPMBP1 (Prenylated Metal Binding Protein 1). Para tal, analisamos a linhagem mutante, a estrutura da proteína prevista, as possíveis relações filogenéticas com outros genes e o padrão de expressão desse gene. Em paralelo, induzimos a mobilização do elemento Ds da sua inserção original no T-DNA da linhagem original para gerar novas linhagens mutantes. A proteína codificada por esse gene contém um motivo de associação a metais pesados e um sítio de prenilação. Identificamos esse gene como um membro de um ramo até agora desconhecido da família das Proteinas Farnesiladas (FP), proteínas preniladas que se ligam a íons metálicos. Nossas análises mostram que a expressão de OsPMBP1 é semelhante em raízes e na porção aérea de plântulas, porém, em condições de excesso de alumínio, a expressão nesses órgãos se altera, sendo induzida em folhas e caules enquanto que em raízes é reprimida. Outros genes da família FP também respondem à estresses por excesso de íons metálicos, e foram caracterizados como diretamente envolvidos no transporte e/ou detoxicação de metais pesados. Em condições normais, esse gene parece ser regulado pelo relógio circadiano, sendo um gene de expressão tardia, com pico de expressão próximo ao entardecer. A análise detalhada da linhagem Ds72 indicou que não há ligação entre o fenótipo observado e a inserção de T-DNA. Os dados obtidos demonstram que OsPMBP1 pode estar envolvido na resposta a estresse por metal pesado ou no transporte desses íons, porém ainda são necessários mais estudos para melhor entender a função desse gene na planta.
The current number of identified genes in the rice genome (Oryza sativa ssp japonica) is about 32,000. Thanks to the efforts of different researchers around the world, the role of many of these genes is currently better understood. The gene function can be inferred mainly by the analysis of its expression pattern, the structure of the encoded protein and by the analysis of mutants where the gene is interrupted, silenced or over-expressed. For that purpose, our research group has developed transgenic lines by the insertion of a T-DNA construction that is based on the two-component iAc/Ds system of transposons. One of these lines shows reduced height and has the T-DNA tag interrupting a locus, whose gene function is yet unknown. The aim of the present study is the identification and characterization of this gene, which we named as OsPMBP1 (Prenylated Metal Binding Protein 1). For that, we analyzed the mutant line, the predicted protein structure, the filogenetic relationship with others proteins, and its expression pattern. In parallel, we induced the mobilization of the Ds element from the original T-DNA insertion to generate new mutant lines. OsPMBP1coded protein has a heavy metal associated domain (HMA) and a prenylation site. We identified this gene as a member of an unknown branch of the Farnesylated Proteins family (FP), composed mostly of prenylated metal binding proteins. Ours analyses showed that the OsPMBP1 expression is equivalent in seedling roots and shoots, but, under aluminum excess, its expression change in these organs, it was inducted in the shoots and repressed in the roots. Others genes of the FP family also respond to metallic ion stresses and have been characterized as directly involved in heavy metal transport and detoxification. In normal conditions, this gene is apparently regulated by the circadian clock, with expression apices close to the evening/dusk interface. The detailed analysis of the Ds72 line indicated that there isn’t a relationship between the observed phenotype and the T-DNA insertion. The data showed that OsPMBP1 may be involved in the response to heavy metal stress or in its transport, but more analyses are required to a better understanding of the role of this gene in the plant.

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34

Leinartaité, Lina. "Zinc in folding and misfolding of SOD1 : Implications for ALS". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-107543.

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing degeneration of upper and lower motor neurons. Most ALS cases are sporadic; only 6% are associated with mutations in Cu, Zn superoxide dismutase (SOD1). It is believed, however, that sporadic and familiar forms of ALS share a common mechanism, where SOD1 plays an important role: SOD1 knockout mice do not develop ALS, whereas the overexpression of human SOD1 in mice produces ALS-like symptoms. Increasing evidence suggest that the SOD1 structure gains cytotoxic properties, but detailed description of the toxic species is missing. This thesis work is focused on understanding how structural and dynamic properties of SOD1 change along its folding free-energy landscape and indicates the structural hot-spots from where the cytotoxic species may originate. Thus, binding of the zinc controls folding, stability and turnover of SOD1: (i) miscoordination of Zn2+ by the Cu-ligands speeds up folding of the SOD1 core structure, however, it stabilizes SOD1 in a state where both active-site loops IV and VII are unfolded, (ii) coordination of Zn2+ in the Zn-site, induces the folding of loop VII and stabilizes the native and functional fold of both active-site loops and (iii) the tremendous stability gain due to Zn-site metallation corresponds to a folded state’s lifetime of > 100 years, thus the cellular lifetime of SOD1 is likely controlled by Zn2+ release, which again is coupled to opening of active-site loops. Hence the active-site loops IV and VII stand out as critical and floppy parts of the SOD1 structure. Moreover, a number of ALS-associated mutations, benign to apo-SOD1 stability, are shown here to affect integrity of active-site loops in holo-SOD1, which, in turn, increases population of SOD1 species with these loops disorganized. Finally, the close relation between SOD1 and Zn2+ can also act in the reverse direction: a perturbed folding free-energy landscape of SOD1 can disturb Zn2+ homeostasis.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

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35

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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36

Kohn, Wayne David. "Studies of electrostatic, salt, and metal-binding interactions in the Ã-helical coiled-coil, a model protein system". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34790.pdf.

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37

Valiveti, Aswani Kumar. "Structural characterization of metal and DNA binding to DREAM protein, a calcium sensing transcriptional repressor in pain modulation". College Park, Md. : University of Maryland, 2006. http://hdl.handle.net/1903/3975.

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Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Molecular and Cell Biology. 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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38

Burkitt, William Ian. "Metal-binding non-covalent protein complexes studied by electrospray ionisation and Fourier transform ion cyclotron resonance mass spectrometry". Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429814.

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39

Wernérus, Henrik. "Engineering of staphylococcal surfaces for biotechnological applications". Doctoral thesis, KTH, Biotechnology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3450.

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The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.

Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.

Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.

Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.

The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.

The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.

Key words:affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices

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40

Wang, Xiaoyang. "Design, Construction and Investigation of Synthetic Devices for Biological Systems". University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1314041031.

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41

Waldron, Kevin. "Analysis of cellular metal pools : the role of periplasmic iron-binding protein FutA2 in copper supply in Synechocystis PCC 6803". Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435565.

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42

Ostendorp, Thorsten. "Structure and function of the metal-binding protein S100B and its interaction with the receptor for advanced glycation end products". [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-23752.

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43

Hoang, Huy Ngoc. "Metal clips for folding peptides : a study of palladium (II) binding to histidine residues in short peptides stabilize (sic) their a-Helical conformation in solutions /". St. Lucia, Qld, 2003. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17478.pdf.

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44

Tait, Timo. "The production and purification of functional steroid hormone receptor ligand binding domains towards the development of a biological endocrine disruptor detection system". Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/96811.

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Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography.
AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

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45

Erlandsson, Lisa-Marie. "Understanding the Involvement of Leukocyte Cell-derived Chemotaxin 2 (LECT2) in Amyloidosis". Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-162631.

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Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.

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46

Suzuki, Noriaki. "Applications of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and x-ray photoelectron spectroscopy (XPS) to study interactions of genetically engineered proteins with noble metal films /". Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/10618.

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47

Moulin, Pauline. "Caractérisation du transporteur de zinc Adc/Lmb de Streptococcus agalactiae". Thesis, Tours, 2017. http://www.theses.fr/2017TOUR3308/document.

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Dans cette étude, le transporteur ABC de zinc de Streptococcus agalactiae, première cause d’infections materno-foetale en France, a été caractérisé. Nous avons montré que ce transporteur se compose, du complexe perméase-ATPase AdcCB, associé à trois protéines membranaires Lmb, AdcA et AdcAII redondantes dans la fixation de zinc. Ce transporteur comporte également deux protéines Sht et ShtII, retrouvées au niveau de la paroi, et nécessaires aux protéines Lmb et AdcAII pour la capture de zinc. L’absence d’un transporteur fonctionnel, par la triple délétion des gènes lmb, adcA et adcAII ou du complexe adcCB, a révélé une inhibition de la croissance et une perturbation de la division de la bactérie lorsqu’elle se trouve dans un environnement carencé en zinc. De plus, nous avons montré que ce transporteur de zinc participe à la survie de la bactérie en milieux biologiques humains, comme le liquide amniotique ou le LCR, où la bactérie est retrouvée lors d’infections, suggérant l’importance du transporteur lors du processus infectieux. Ces résultats ont mis en évidence, pour la première fois, que le zinc assure des fonctions biologiques vitales pour S. agalactiae et que, dans des conditions de forte carence en zinc, le transporteur Adc/Lmb représente le principal système d’acquisition de zinc de la bactérie
In this study, the zinc-ABC transporter of Streptococcus agalactiae, the first cause of materno-foetal infections in France, was characterized. We showed that this transporter is composed of an AdcCB permease-ATPase complex in association with three membrane-associated proteins Lmb, AdcA and AdcAII, which are redundant in zinc-binding. This transporter also possesses two proteins Sht and ShtII, which are associated to the cell wall, and that are necessary for the Lmb and AdcAII proteins for zinc capture. The absence of a functional transporter, by the triple deletion of the lmb, adcA and adcAII genes or the adcCB complex, revealed a growth inhibition and a disruption of the division of the bacterium when it is in a zinc-restricted environment. Furthermore, we showed that the zinc-ABC transporter contributes to the survival of the bacterium in human biological fluids, as the amniotic fluid or the cerebrospinal fluid, where the bacterium is found during infections, suggesting the importance of the transporter during the infectious process. These results hightlighted, for the first time, that zinc has biologically vital functions in S. agalactiae and that, under high zinc deficiency conditions, the Adc/Lmb transporter is the main zinc acquisition system of the bacterium

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48

Elison, Kalman Grim. "Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea". Thesis, Uppsala universitet, Strukturbiologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-387943.

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This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR.

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49

Lin, Yu-Feng y 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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50

SHARMA, SHAILESH. "Bioinformatics of metal binding proteins and genome wide analysis". Doctoral thesis, 2009. http://hdl.handle.net/2158/485462.

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Tesis sobre el tema "Metal-binding proteins"

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