[NiFe] hydrogenases are found in a variety of microorganisms and catalyze the reversible oxidation of hydrogen gas to protons and electrons. This enzyme has generated intense interest due to its contribution to pathogenicity in certain organisms as well as its application in bioremediation and the production of hydrogen as an alternative fuel source. The biosynthesis of the dinuclear active site requires a number of accessory proteins to chaperone and insert the metal cofactors to the awaiting large subunit of hydrogenase. The proteins responsible for nickel delivery to Escherichia coli hydrogenase 3 are HypA, HypB, and SlyD, however the mechanism by which this is accomplished is unclear. The goal of this work was to analyze the metal-binding abilities and protein interactions of these nickel insertion proteins to enhance our understanding of their roles. Isolated N-terminal peptide of HypB has similar high-affinity metal-binding to the full-length protein. This peptide binds nickel in a square planar site with three cysteinyl and a fourth N-terminal amine ligand. Additionally, studies with SlyD and HypA reveal protein interactions that occur during hydrogenase maturation. Pull-down experiments of a tagged variant of hydrogenase revealed multi-protein complexes with HypA, HypB, and SlyD. A complex between SlyD and hydrogenase forms prior to both nickel and iron insertion, supporting chaperone activity of SlyD during hydrogenase maturation. HypA can interact with hydrogenase in the absence of HypB and SlyD, and a possible role as the bridging protein during the nickel insertion event is proposed. In addition, fluorescent imaging of E. coli cells using a fluorescently labeled streptavidin conjugate revealed localization of both Strep-tagged II hydrogenase and HypA at or near the cell membrane, suggesting that enzyme maturation occurs proximal to metal transporters. This work provided a deeper understanding of the role that each of these proteins play in [NiFe] hydrogenase assembly and is helpful for any future applications of this enzyme.

Thesis (Ph.D.)--Michigan State University. Cell and Molecular Biology Program, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.

Orientador: Pedro de Magalhães Padilha / Banca: Marcos Roberto de Mattos Fontes / Banca: Lucilene Delazari dos Santos / Resumo: Neste trabalho de Dissertação, buscou-se avaliar as metaloproteínas presentes em amostras de colostro, leite de transição e leite maduro humanos. Utilizando-se técnicas clássicas da proteômica - eletroforese bidimensional, espectrometria de massas e bioinformática - e uma técnica clássica de determinação de metais - a absorção atômica - foi possível identificar e avaliar a ligação de cálcio, ferro e zinco a algumas proteínas do leite materno. Essa nova área de estudo, denominada metaloproteômica, procura compreender o papel dos íons metálicos em sistemas biológicos, mais especificamente o seu papel em relação às proteínas. Três grupos de proteínas foram identificados: soroalbumina, IgA secretória (sIgA) e -caseína. Encontrou-se cálcio e ferro ligados à sIgA, cálcio e zinco ligados à soroalbumina, e cálcio, ferro e zinco ligados à -caseína. As peculiaridades dessas interações são discutidas no texto. Além disso, analisou-se as mudanças na expressão dessas proteínas do decorrer da lactação com base nas imagens dos géis de poliacrilamida. A sIgA esteve presente em maiores concentrações no colostro, a -caseína foi mais expressa no leite de transição e maduro e não houve alteração na expressão da soroalbumina / Abstract: At this present work, the goal was to evaluate metalloproteins in samples of human colostrum, transitional milk and mature milk. Making use of classical techniques of proteomics - two-dimensional electrophoresis, mass spectrometry and bioinformatic tools - and a classic technique of metal detrmination - atomic absorption - it was possible to identify and evaluate the binding of calcium, iron and zinc to some human milk proteins. This new area of study, named metalloproteomics, seeks the understanding of the role of metal ions in biological systems, mostly proteins. Three groups of proteins were identified: serum albumin, secretory IgA (sIgA) and -casein. It was found calcium and iron bound to sIgA, calcium and zinc bound to serum albumin and calcium, iron and zinc bound to -casein. The specialties of these interactions are discussed in the text. Besides, the changes in these proteins expressions, based in gel analysis, were evaluated. sIgA presented higher concentrations in colostrum, -casein in transitional and mature milk, and serum albumin presented no alterations / Mestre

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-01Bitstream added on 2014-06-13T18:50:16Z : No. of bitstreams: 1 000736006.pdf: 2441606 bytes, checksum: d444cf05acb64b49dc0cbdbb11978c77 (MD5) / Neste trabalho de Dissertação, buscou-se avaliar as metaloproteínas presentes em amostras de colostro, leite de transição e leite maduro humanos. Utilizando-se técnicas clássicas da proteômica - eletroforese bidimensional, espectrometria de massas e bioinformática - e uma técnica clássica de determinação de metais – a absorção atômica – foi possível identificar e avaliar a ligação de cálcio, ferro e zinco a algumas proteínas do leite materno. Essa nova área de estudo, denominada metaloproteômica, procura compreender o papel dos íons metálicos em sistemas biológicos, mais especificamente o seu papel em relação às proteínas. Três grupos de proteínas foram identificados: soroalbumina, IgA secretória (sIgA) e -caseína. Encontrou-se cálcio e ferro ligados à sIgA, cálcio e zinco ligados à soroalbumina, e cálcio, ferro e zinco ligados à -caseína. As peculiaridades dessas interações são discutidas no texto. Além disso, analisou-se as mudanças na expressão dessas proteínas do decorrer da lactação com base nas imagens dos géis de poliacrilamida. A sIgA esteve presente em maiores concentrações no colostro, a -caseína foi mais expressa no leite de transição e maduro e não houve alteração na expressão da soroalbumina / At this present work, the goal was to evaluate metalloproteins in samples of human colostrum, transitional milk and mature milk. Making use of classical techniques of proteomics – two-dimensional electrophoresis, mass spectrometry and bioinformatic tools – and a classic technique of metal detrmination – atomic absorption – it was possible to identify and evaluate the binding of calcium, iron and zinc to some human milk proteins. This new area of study, named metalloproteomics, seeks the understanding of the role of metal ions in biological systems, mostly proteins. Three groups of proteins were identified: serum albumin, secretory IgA (sIgA) and -casein. It was found calcium and iron bound to sIgA, calcium and zinc bound to serum albumin and calcium, iron and zinc bound to -casein. The specialties of these interactions are discussed in the text. Besides, the changes in these proteins expressions, based in gel analysis, were evaluated. sIgA presented higher concentrations in colostrum, -casein in transitional and mature milk, and serum albumin presented no alterations

Two separate systems were developed for the expression of recombinant metalloproteins. A synthetic gene encoding the lipase-solubilized form of bovine liver microsomal cytochrome b₅ was designed and assembled for expression in E. coli. Analysis of the initial recombinant cytochrome revealed differences in several physical characteristics of the molecule compared to the authentic bovine liver species, including a reduction potential that was lower by 17 mV. Further studies showed the primary sequence of the initial recombinant differed from the authentic protein in the amidation status of three residues which, when corrected yielded a recombinant protein identical in behaviour to the authentic protein. The participation of Ser64 in the stabilization of the oxidized form of cytochrome b₅ was investigated using site-directed mutagenesis to alter this residue to Ala, which was predicted to ablate a hydrogen bond formed between the protein and heme-propionate 7. Spectroelectrochemical analysis of this variant showed that the reduction potential had been shifted downwards by 7 mV, in contrast to predictions from a structural model describing the red/ox behaviour of cytochrome b₅ (Argos and Mathews, 1975). The role of heme carboxylates in determining the reduction potential was confirmed for both the wild-type and Ala64 variants by heme replacement studies using the esterified derivative of protoporphyrin IX, suggesting that the presence of free carboxylates contributes to the stabilization of the oxidized species. In addition, constructions for the expression of the trypsin-solubilized form of bovine liver microsomal cytochrome b₅ and the erythrocytic form of human cytochrome b₅ are described. A tissue culture cell system was developed for the expression of the N-terminal half molecule of human serum transferrin. The recombinant molecule (hTF/2N) was secreted at high levels from selected eukaryotic cells, and displayed high identity with the proteolytically-derived molecule from authentic human serum transferrin as judged by sequence analysis, electrophoretic mobility and iron binding capacity. A construction for the expression of the C-terminal half molecule was assembled but failed to express recombinant protein when introduced into tissue culture cells. The production of these two heterologous expression systems allows for high-level recovery of recombinant protein and provides a convenient approach to structure-function studies employing site-directed mutagenesis techniques. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology

La nitrate réductase périplasmique de Rhodobacter sphaeroides catalyse la réduction du nitrate en nitrite. C'est une métalloenzyme qui comprend un cofacteur à molybdène, un centre fer - soufre et deux hèmes.La réactivité du cofacteur à molybdène reste mal comprise pour plusieurs raisons. Entre autres : l'hétérogénéité des signatures RPE Mo(V), état semi-réduit du site actif, et l'existence d'états inactifs de l'enzyme selon les conditions.Pour comprendre la réactivité et la pertinence catalytique des principales espèces Mo(V), nous avons entrepris une caractérisation des processus d'activation et d'inactivation par électrochimie sur film de protéines, et une étude de leur structure par spectroscopies RPE et HYSCORE.Nos observations cinétiques suggèrent que l'activation irréversible de l'enzyme implique un réarrangement d'une des ptérines du cofacteur à Mo.Ceci est mis en évidence par la modification des couplages magnétiques intercentres du fait de l'activation, et par des modifications de structure au delà de la première sphère de coordination du Mo.Enfin, l'étude de l'inactivation réversible de l'enzyme par électrochimie montre l'implication des différents états redox du site actif dans le mécanisme d'inhibition, et donne les conditions nécessaires au piégeage de formes Mo(V) actives. / Rhodobacter sphaeroides periplasmic nitrate reductase catalyses the reduction of nitrate into nitrite. It is a metalloenzyme containing a molybdenum cofactor, an iron - sulfur cluster, and two haems.The reactivity of the molybdenum cofactor remains elusive for many reasons. Among others : the heterogeneity of the EPR signatures of Mo(V), the semi-reduced state of the active site, and the existence of inactive states of the enzyme, depending on conditions.In order to understand the reactivity and the catalytic relevance of the major Mo(V) species, we have undertaken a characterisation of the activation and inactivation processes by protein-film-electrochemistry, and a study of their structure by EPR and HYSCORE spectroscopies.Our kinetic observations suggest that the irreversible activation of the enzyme involves a rearrangement of one of the pterins of the Mo cofactor.This is evidenced by the modification of intercentre magnetic couplings due to the activation, and by structural modifications beyond the first coordination sphere of Mo.Finally, the study of enzyme reversible inactivation by electrochemistry shows the involvement of the different redox states of the active site in the inhibition mechanism, and yields the necessary conditions to trapping active Mo(V) forms.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 15.

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.

Thesis (Ph.D.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.