Marburg, Universiẗat, Diss., 2003. / Erscheinungsjahr an der Haupttitelstelle: 2002.

The Crystal Structure of Sl Nuclease from Aspergillus oryzae S 1 nuclease from Aspergillus oryzae is a glycoprotein of 32 kDa molecular weight. The protein has two enzymatic activities: it is an endo-exonuclease with high specificity for single stranded nucleic acids, and it has an additional 3' -nucleotidase activity. S 1 nuclease is widely used in molecular biology as a single-strand specific nuclease due to its high stability and efficiency. It cleaves single-stranded regions of nucleic acids producing 5' -nucleotides without significant side-reactions. The crystal structure of S 1 nuclease has been determined to 1.7 A resolution by molecular replacement based on the known structure of PI nuclease from Penicillinum citrinum, which has 49 % sequence identity compared to S 1. The overall fold and the active site of S 1 nuclease is basically identical to that of PI nuclease, and also very similar to Phospholipase C from Bacillus cereus and alpha-toxin from Clostridium perfringens. The characteristic feature of this family of enzymes is a trinuclear zinc cluster in their active sites. A BLAST search in the sequence databases revealed several other protein sequences from bacteria, protozoa and plants possessing an approximately 30 % sequence identity compared to S 1 nuclease, but showing an almost complete conservation of structurally and functionally important residues. Soaking and co-crystallisation experiments with substrate analogues have been carried out in order to obtain an enzyme-substrate complex. These efforts have not resulted in the structure determination of any complexes under crystallisation conditions: no binding of substrate has been observed. Nevertheless, an enzyme mechanism has been proposed based on structural data of S 1 nuclease and nucleases with similar active sites. The Crystal Structure of an Sm-Related Protein from Archaeoglobus fulgidus In eukaryotes Sm and Sm-like proteins are the core components of the small nuclear ribonucleoprotein particles (snRNPs), which are involved in a variety of functions including rRNA processing, tRNA maturation and pre-mRNA processing. The Sm proteins are 70 to 120 amino acids long and share a common bi-partite signature sequence. The spliceosome, where the transesterification reaction of splicing occurs, is assembled by several snRNPs named after their constituting snRNA: U1, U2, U4, U5 and U6. An snRNA contains a short single stranded, uridine rich sequence motif, where the Sm proteins bind, but the three-dimensional arrangement of the Sm proteins and the mode of binding is unknown. In humans there are seven different canonical Sm proteins, which according to biochemical and electron microscopic studies seem to form a seven membered ring in vitro. Recently two crystal structures of human Sm protein dimers have been published. Interestingly Sm-related protein sequences have been found in the available genomic database of various Archaebacteria based on sequence homology. In contrast with eukaryotes only one or two Sm-related protein sequences have been identified in one organism. Their function is currently unknown, since analogous pre-mRNA splicing does not occur in Archaebacteria. Two Sm-related proteins of Archaeoglobus fulgidus have been cloned and expressed as fusion proteins. One of them called AF-Sm2 has been o crystallised utilising ammonium sulphate as precipitant and solved to 1.95 A resolution by SIRAS using a single mercury derivative. AF-Sm2 crystallises in a hexagonal space group (P6) and contains one molecule per asymmetric unit. The 77 residue long protein has a very similar fold compared to the solved human Sm protein structures: a short N-terminal a-helix followed by a five stranded, strongly bent, U-shaped ~-sheet resulting in a barrel-like overall fold. Six AF-Sm2 molecules form a ring in the crystal structure mediated by extensive hydrophobic and hydrogen-bonding interactions. Gel filtration experiments have indicated a pH dependence of oligomerisation in accordance with the crystallisation experiences. Currently the target of the Sm-related proteins of Archaeoglobus fulgidus and the stochiometry of oligomerisation in vivo is completely unknown.

Copper is an essential biological metal. Many enzymes such as cytochrome C oxidase and superoxide dismutase use copper as a prosthetic group. However, free copper can be harmful by generating reactive oxygen species through Fenton reactions (Linder and Hazegh-Azam 1996). Therefore, copper is tightly regulated throughout the cell. Cu+ ATPases are transporters that regulate Cu+ homeostasis. Mutations to these Cu+ ATPases can cause diseases (Wilson's and Menkes diseases) (Ferenci 2005). CopA from Archaeoglobus fulgidus is a model Cu+ ATPase; it contains eight transmembrane helices and a cytosolic ATP binding domain. CopA is assisted by a Cu+ chaperone (CopZ) which we have shown delivers Cu+ to CopA via protein-protein interactions (Guerrero-Guerrero and Arguello 2008). CopZ has two domains which are both able to bind Cu+ (Guerrero-Guerrero and Arguello 2008). The N-domain has a mononuclear Zn2+ site, a [2Fe-2S] cluster and is extremely rich in cysteines (Sazinsky, LeMoine et al. 2007). To explore the role of cysteines and their Cu+ binding capacity, each cysteine was mutated to serine. All resulting proteins were able to activate CopA ATPase. However, mutation of two Cysteines (Cys4 and Cys38) led to binding of two additional Cu+ ions. Since these two Cys participate in Zn2+ coordination we hypothesized that the coordination of Cu+ might take place with the displacement of Zn2+. In order to explore this hypothesis, CopZ-WT was incubated with an excess amount of Cu+, followed by atomic absorption to measure the presence of Zn2+. These experiments demonstrate a displacement of Zn2+ by Cu+. Thus, our results show that the second Cu+ binding site in A. fulgidus CopZ is constituted by the same Cysteines participating in Zn2+ binding.

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Based on what we have found, we proposed a model for Ni presenting by HypB involved in hydrogenase maturation. HypB binds two Ni ions in the apo- and GDP-bound form. Ni binding also induces dimerization of HypB. Upon GTP binding, HypB can bind an extra Ni ion at the dimeric interface. GTP hydrolysis will release the extra Ni ion, which may be subsequently inserted into hydrogenases during the maturation process. / Furthermore, two Ni binding sites were determined in a monomeric HypB. One is the cluster including C92, H93 and C122, the other is composed of H97 and H101. Upon GTP-dependent dimerization, HypB can bind an extra Ni ion. Our results have shown that the C92/H93/C122 is involved in binding the extra Ni ion, and such binding requires both cysteine residues in the reduced form. Since the GTP-induced dimerization of HypB is coupled to bind an extra Ni, so HypB could act as a GTP-mediated switch that regulate one Ni release from the GTP-bound form to the GDP-bound form. / In the future, we will attempt to crystallize AfHypB in complex with GDP, GTP analogue and AfHypA. Availability of good quality crystals will pave way for the structure determination of AfHypA and AfHypA/HypB complex. And the results obtained will provide a better understanding of the mechanism of functional interaction between HypA and HypB and how HypA and HypB play a role in Ni ion delivery for hydrogenase maturation. / The assembly of the [NiFe]-hydrogenases requires incorporation of Ni ions into the enzyme's metallocenter, which process requires the GTPase activity of HypB and HypA. Due to the essential role in assembly of the active site of hydrogenases, the two proteins were defined as hydrogenase maturation factors. To better understand the mechanism of GTP hydrolysis-dependent Ni delivery accomplished by HypB and HypA, our work focuses on the structure-function study of AfHypB from Archaeoglobus fulgidus and the functional interaction between AfHypA and AfHypB. / The intrinsic GTPase activity of AfHypB is very low, suggesting that AfHypB requires a G-protein activating protein (GAP) to activate its GTPase activity. Although AfHypB can interact with AfHypA to form 1:1 heterodimer, our data suggests that AfHypA is not a GAP for AfHypB. In addition, the FRET results showed that AfHypA could serve as a GEF (G-protein exchange factor) to activate the AfHypB from GDP-bound form to GTP-bound form and facilitate the dissociation of AfHypB dimer in the presence of GMPPNP. / Up to now, we have solved the structure of apo-AfHypB by X-ray crystallography. Crystals of AfHypB were grown using the hanging-drop-vapor-diffusion method and diffracted to ∼2.3 A. It belonged to space group P2(1)2(1)2, with unit cell dimensions a=72.49, b=82.33, c=68.66 A, alpha=beta=gamma=90°. Two molecules of AfHypB were found in an asymmetric unit. Structural comparison between the apo-AfHypB and GTP-bound HypB from M jannachii showed that the GTP binding broke the salt-bridge between K43 and D66, and induced conformational changes in the switch I loop and helix-3, which caused the HypB to form dimer and bind an extra Ni ion. The GTP-bound form of HypB was ready for Ni presenting. And GTP hydrolysis could induce the conformational changes of HypB in the switch I loop and helix-3, which dissociate the HypB dimer into the monomeric GDP-bound form. / Li, Ting. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 98-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.