Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 147-155). Also available for download via the World Wide Web; free to University of Oregon users.

Universiẗat, Diss., 2001--Köln.

Universiẗat, Diss., 2002--Göttingen.

Chan, Kwok Ho. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgements --- p.II / Abstract --- p.III / 摘要 --- p.IV / Table of Contents --- p.V / Abbreviations --- p.XIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- GTPase in general --- p.1 / Chapter 1.2 --- G proteins and GTP switch --- p.2 / Chapter 1.3 --- Structural similarities in GTPase --- p.3 / Chapter 1.4 --- G proteins in bacteria --- p.3 / Chapter 1.5 --- Background information of the protein family EngB --- p.4 / Chapter 1.6 --- Basic information of EngB in Thermotoga maritima --- p.5 / Chapter 1.7 --- Objectives of this work --- p.6 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemical reagents --- p.8 / Chapter 2.1.2 --- Buffers / Chapter 2.1.2.1 --- Preparation of buffers --- p.10 / Chapter 2.1.2.2 --- Buffers for common use --- p.11 / Chapter 2.1.3 --- Expression strains and plasmids --- p.14 / Chapter 2.1.4 --- Primer list --- p.14 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Preparation of competent cells --- p.15 / Chapter 2.2.2 --- Cloning / Chapter 2.2.2.1 --- Cloning of target genes by PCR --- p.15 / Chapter 2.2.2.2 --- Agrose gel electrophoresis --- p.17 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gel --- p.17 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.18 / Chapter 2.2.2.6 --- Transformation and plating out transformants for miniprep --- p.19 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.20 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.21 / Chapter 2.2.2.9 --- Confirmation of miniprep product by restriction enzyme digestion..… --- p.22 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.23 / Chapter 2.2.3 --- Expression of the recombinant MBP-TM EngB protein and SBP-CBP EC EngB / Chapter 2.2.3.1 --- Transformation for protein expression --- p.23 / Chapter 2.2.3.2 --- Preparation of starter culture --- p.24 / Chapter 2.2.3.3 --- Expression of recombinant protein --- p.24 / Chapter 2.2.3.4 --- Cell harvesting --- p.24 / Chapter 2.2.3.5 --- Releasing the cell content --- p.25 / Chapter 2.2.3.6 --- Check for protein expression by SDS-PAGE --- p.25 / Chapter 2.2.4 --- Purification of TM EngB / Chapter 2.2.4.1 --- SP ion-exchange chromatography --- p.27 / Chapter 2.2.4.2 --- Thrombin digestion to remove MBP tag --- p.28 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.29 / Chapter 2.2.4.4 --- Gel filtration chromatography --- p.29 / Chapter 2.2.5 --- Purification of SBP-CBP EC EngB / Chapter 2.2.5.1 --- SP ion-exchange chromatography --- p.30 / Chapter 2.2.5.2 --- Gel filtration chromatography --- p.31 / Chapter 2.2.6 --- Protein concentration quantitation --- p.32 / Chapter 2.2.7 --- Crystallography of TM EngB / Chapter 2.2.7.1 --- Crystallization preparation --- p.32 / Chapter 2.2.7.2 --- Crystallization screening by sitting drop method --- p.32 / Chapter 2.2.7.3 --- Optimization of crystallization conditions --- p.33 / Chapter 2.2.7.4 --- X-ray diffraction --- p.33 / Chapter 2.2.8 --- Thermodynamics studies of proteins / Chapter 2.2.8.1 --- Preparation of protein sample --- p.34 / Chapter 2.2.8.2 --- Guanidine-induced denaturation experiment --- p.34 / Chapter 2.2.8.3 --- Thermal-induced denaturation experiment --- p.35 / Chapter 2.2.9 --- Binding assay to study affinity for ligands --- p.36 / Chapter 2.2.9.1 --- Using GDP analogue mant-GDP to detect formation of enzyme-ligand complex (TM EngB-mant-GDP) --- p.36 / Chapter 2.2.9.2 --- Basic information of Fluorescence spectroscopy --- p.36 / Chapter 2.2.9.3 --- Determination of λem and λex --- p.37 / Chapter 2.2.9.4 --- Studying ligand affinity by titration with ligand analogue --- p.37 / Chapter 2.2.10 --- Pull down experiment to study interacting partner of E. coli EngB --- p.38 / Chapter 2.2.10.1 --- Preparing protein extracts from E. coli --- p.38 / Chapter 2.2.10.2 --- Preparing streptavidin resin --- p.39 / Chapter 2.2.10.3 --- Binding of dual-tagged E. coli EngB to streptavidin resin --- p.39 / Chapter 2.2.10.4 --- Purifying protein using the prepared streptavidin resin --- p.40 / Chapter 2.2.10.5 --- Preparing calmodulin resin --- p.41 / Chapter 2.2.10.6 --- Binding of dual-tagged E.coli EngB to calmodulin resin --- p.41 / Chapter 2.2.10.7 --- Analysis of dual-tag affinity purified protein --- p.42 / Chapter 2.2.11 --- Silver staining of acrylamide gel / Chapter 2.2.11.1 --- Staining reagents --- p.42 / Chapter 2.2.11.2 --- Staining procedures --- p.43 / Chapter Chapter 3 --- Structure determination of T. maritima EngB by X-ray crystallography / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Generation of TM EngB expression construct --- p.45 / Chapter 3.3 --- Expression and purification of TM EngB --- p.46 / Chapter 3.4 --- TM EngB was crystallized with freshly purified TM EngB --- p.47 / Chapter 3.5 --- Data processing of diffraction data and structure refinement of TM EngB …… --- p.48 / Chapter 3.6 --- Apo-form TM EngB was obtained by unfolding and refolding --- p.49 / Chapter 3.7 --- Crystallization of apo-form TM EngB --- p.50 / Chapter 3.8 --- Data processing of diffraction data and structure refinement of apo-form TM EngB --- p.51 / Chapter 3.9 --- Producing EngB-GDP complex crystal from apo-from EngB --- p.52 / Chapter 3.10 --- TM EngB is a monomer in solution --- p.54 / Chapter 3.11 --- Summary of chapter three --- p.55 / Tables and figures of chapter three --- p.57 / Chapter Chapter 4 --- Structural details of TM EngB / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Overall fold of TM EngB --- p.67 / Chapter 4.3 --- Mode of nucleotide binding of TM EngB --- p.68 / Chapter 4.4 --- Structural differences in switch I region between chain A and chain B in crystal structure of TM EngB/GDP complex --- p.70 / Chapter 4.5 --- Structural difference between TM EngB/GDP complex and apo TM EngB --- p.73 / Chapter 4.6 --- Summary of chapter four --- p.73 / Tables and figures of chapter four --- p.76 / Chapter Chapter 5 --- Purified TM EngB is Active for binding guanine nucleotide but inactive for GTPase hydrolysis activity / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Studying ligand affinity by competitive binding experiment --- p.88 / Chapter 5.3 --- GDP binds to TMEngB with higher affinity than GTPyS --- p.91 / Chapter 5.4 --- TM EngB showed very low intrinsic GTPase activity --- p.92 / Chapter 5.5 --- Discussion --- p.93 / Tables and figures of chapter five --- p.95 / Chapter Chapter 6 --- Thermostability of EngB of T. maritima / Chapter 6.1 --- Introduction --- p.98 / Chapter 6.2 --- Guanidine hydrochloride - induced unfolding --- p.98 / Chapter 6.3 --- Thermal-induced unfolding --- p.99 / Chapter 6.4 --- Structural comparison of thermophilic and mesophilic EngB --- p.100 / Chapter 6.5 --- Discussion --- p.102 / Tables and figures of chapter six --- p.105 / Chapter Chapter 7 --- Construction of a dual-tag affinity pull-down system for finding interacting partner of EngB / Chapter 7.1 --- Introduction --- p.112 / Chapter 7.2 --- Preparation of dual-tagged E.coli EngB / Chapter 7.2.1 --- Cloning of SBP-CBP-EC EngB expression construct --- p.113 / Chapter 7.2.2 --- Expression and purification of SBP-CBP-EC EngB --- p.114 / Chapter 7.3 --- Pull down using dual tagged E.coli EngB as bait to isolate potential interacting partners of EngB --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Tables and figures of chapter seven --- p.117 / Chapter Chapter 8 --- Conclusion --- p.122 / References --- p.124

University, Diss., 2007--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

La bactérie hyperthermophile Thermotoga maritima a été cultivée dans un fermenteur dans lequel la concentration en O2 a été rigoureusement contrôlée. A 80°C et pH 7, il a été démontré que T. maritima pouvait survivre à des expositons de durées variables à l’O2 et qu’elle était capable de le consommer. La vitesse spécifique de consommation de l’O2 a été estimée à 73.6 µmoles O2.min-1.g protéines-1 lors d’une courte exposition à l’O2 (30 min). De longues expositions à l’O2 (20 h) nous ont permis de démontrer que la présence d’O2 ralentissait la croissance de T. maritima et conduisait à un shift du métabolisme vers la production de lactate aux dépens de l’acétate et à un arrêt de production d’H2. Dans ces conditions, il a été constaté que 73% du glucose était consommé selon un métabolisme partiellement oxydatif faisant intervenir simultanément les deux voies Embden-Meyerhof et Entner-Doudoroff de la glycolyse. En l’occurrence, l’oxydation incomplète du glucose est corrélée à la réduction de l’O2 en eau. Les études transcriptomiques ont montré que cette réduction de l’O2 résultait d’une cascade de réactions intermédiaires faisant intervenir des enzymes de type peroxydases [activation de l’expression des enzymes Ahp (alkyl hydroperoxyde réductase), Bcp1 et Bcp2 (thiol peroxydase thioredoxin-dépendante)] qui acheminent les électrons libérés via les radicaux libres. D’autres enzymes comme la rubréryhtrine et la neelarédoxine interviendraient pour détoxiquer les espèces réactives d’O2. Les électrons libérés seraient au final utilisés pour réduire l’O2 en H2O par l’enzyme FprA, dont l’expression varie en fonction du potentiel redox du milieu de culture. Ce schéma est proposé comme un des éléments essentiels du dispositif enzymatique permettant la consommation de l’O2 et la protection des cellules contre les effets des espèces réactives de l’oxygène chez T. maritima. / Batch cultures of the hyperthermophilic bacterium Thermotoga maritima were performed in a bioreactor where O2 concentrations in the gas phase were strictly controlled. At 80°C and pH 7, we demonstrated that T. maritima survived despite being exposed to oxygen at different times and that it consumed it. O2 uptake rate was estimated at 73.6 µmoles O2 min-1g proteins-1 during a short exposure to O2 (30 minutes). A long time exposure of T. maritima cultures to oxygen (20h) led to a drastic reduction in growth, together with a shift in glucose metabolism towards lactate instead of acetate production and a stop in H2 production. Under these conditions, it has been observed that 73% of glucose was partially oxidised by using both Embden-Meyerhof and Entner-Doudoroff glycolytic payhways. Uncomplete oxidation of glucose is correlated to a reduction of O2 to H2O. Transcription analyses revealed that this reductive process of O2 involved enzymes like peroxidases [activation of alkyl hydroperoxide reductase (ahp), bcp1 and thioredoxin-dependent thiol peroxidase (bcp 2)]. Moreover, genes encoding reactive oxygen species (ROS)-scavenging systems (neelaredoxin and rubrerythrin), were found to be upregulated during oxygen exposure. The oxygen reductase FprA, which expression was shown to depend on the redox level of the culture medium, is proposed as a primary consumer of O2. All these enzymes are essential for T. maritima to consume O2 consumption and to fight against the toxic effects of ROS in cells.

There are large amount of cellulose accumulated in radioactive waste and radioactive pollution sites. It is difficult to clean up these cellulose. In general, waste treatment process can only proceed until the radiation decay to a safty level. Since most cellulolytic microorganisms could not survive in radioactive waste, the accumulation of cellulose in radioactive waste become a serious problem. Deinococcus radiodurans is highly resistant to radiation, UV light, and dryness. It is possible to use this bacterial strain in the bioremediation of radioactive waste. In this study, we found out that there was not much difference on the growth of this organism under radiation and UV light. Cellulose enzyme activity was inhibited by UV irradiation, but not by 32P radiation. The addition of D. radiodurans whole cells or its cell crude extracts could protect the cellulase from UV damage. We also successfully constructed two plasmids, that contained a cel A gene isolated from Thermotoga maritima. These two plasmids had been used to transform Escherichia coli BL21 and D. radiodurans. All transformed bacterial strains could express celA activity. The celA activities in these transformed D. radiodurans strains were not affect by UV irradiation. However, celA enzyme activity in the transformed E. coli was greatly inhibited by UV irradiation up to 78%. Hopefully these two transformed D. radiodurans bacterial strains can be applied to the bioremediation of radioactive waste.

The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates. / Chemistry

No description available.

Thermal stability of theG37 tRNA methyltransferase proteins from Thermotoga maritima and Aquifex aeolicus have been compared using Differential Scanning Calorimetry. It was shown that the Thermotoga protein is remarkably stable and is denatured at temperatures in excess of 100 degrees Centigrade. The Aquifex aeolicus protein was less stable, denaturing broadly at temperatures between 55oC and 100oC. In contrast, the mesophilic E. coli protein was completely denatured at 55oC. Enzymatic activity of the proteins was measured at various temperatures. Both the Thermotoga and Aquifex enzymes are active at ambient temperatures, and display a significant decrease in activity when the temperature is raised above 50oC. This may relate to subtle changes in protein structure causing an effect on the tRNA based assay. Both enzymes contain inter subunit disulfide bonds which might contribute to thermal stability. Assays of the enzymes in the presence of high concentrations of Dithiothreitol (DTT) did not significantly reduce activity at higher temperatures, but did stimulate activity at lower temperatures. Site directed mutagenesis of non -conserved protein sequences within Thermotoga maritima were initiated in order to determine what structures might confer heat stability on the protein. Alanine mutagenesis of lysine residues 103,104 led to reduced catalytic activity, but did increased activity at higher temperatures. Aspartate is the most common residue at the relative position 166 in the variable loop of most TrmD genes. It has been shown that in E. coli this is essential for catalytic activity and possibly the residue which carries out N1 deprotonation on residue G37 in tRNA. In Thermotoga glutamate is present at this position. Alanine mutagenesis of this residue did not eliminate activity suggesting another nearby residue may function in this capacity in the Thermotoga TrmD protein.