No description available.

No description available.

No description available.

No description available.

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

No description available.

<p>Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. </p><p>Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. </p><p>Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.</p>

Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.

is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.