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The chloride intracellular channel (CLIC) family of proteins are an unusual class of chloride channels that possess the ability to auto-insert into cellular membranes. The CLICs exhibit ubiquitous tissue and cellular distributions and adopt a glutathione S-transferase fold in the soluble form that is highly conserved in vertebrates. CLIC homologues have been identified in the model organisms C. elegans and D. melanogaster, and in the former case have been extensively characterised in regards to function. In this thesis, we present the crystal structure of the D. melanogaster CLIC, revealing several unique features in the conserved invertebrate CLIC fold including an elongated C-terminal extension and metal binding site. The bound metal is identified as the potassium cation, resolving concerns regarding previously published work that assign the metal as the isoelectronic calcium cation. It has been reported that a human CLIC protein, CLIC4, translocates to the nucleus in response to cellular stress, facilitated by a putative CLIC4 nuclear localisation signal (NLS). The CLIC4 NLS adopts α-helical structure in the native CLIC4 structural fold. It is proposed that CLIC4 is transported to the nucleus via the classical nuclear import pathway after binding the import receptor, importin-α. We have determined the X-ray crystal structure of a truncated form of importin-α bound to a CLIC4 NLS bearing peptide. The NLS peptide binds the major binding site in an extended conformation similar to that observed for the classical SV40 large T-antigen NLS. A tyrosine residue within the CLIC4 NLS makes surprisingly favourable interactions by forming side chain hydrogen bonds to the importin-α backbone. This structural evidence supports the hypothesis that CLIC4 translocation to the nucleus is governed by the importin-α nuclear import pathway, providing it can undergo a conformational rearrangement that exposes the NLS in an extended conformation. We further analyse importin-α:NLS binding interactions by solving high resolution structures of truncated importin-α containing an empty binding site and bound to the SV40 NLS. A surprising interaction is discovered between importin-α and an NLS-like motif in the endogenous E. coli 30S ribosomal subunit S21, revealing new insight into importin-α recognition of full length cargo.

Phosphorylation/dephosphorylation is likely the most crucial chemical reaction taking place in all living organisms. It is the basis for the regulatory control of many diverse biological events triggered by extracellular effectors. Moreover, it is a ubiquitous element of intracellular signal transduction pathways that regulates a wide range of processes. While protein phosphorylation has been extensively characterized in eukaryotes, far less is known about its emerging counterparts in prokaryotes. This study involved determination of the crystal structures and functional characterization of two protein kinases, YihE and AceK (also a protein phosphatase), and two nucleotide pyrophosphatases, YjjX and YhdE. X-ray crystallographic structure determination combined with bioinformatics analyses, mutageneses and biochemical experiments, both in vitro and in vivo, were utilized for the functional characterization of each protein. YihE was found to be a previously unknown kinase component of a new type of bacterial phospho-relay mechanism, thus adding kinase activity as another response to the Cpx sensing system that functions to maintain cellular homeostasis. AceK, which possesses both kinase and phosphatase activities, modifies isocitrate dehydrogenase (ICDH) to regulate the flux of isocitrate into the glyoxylate cycle. The structures of Acek alone and in complex with its substrate, ICDH, provided us with information to explain the mechanisms underlying its bifunctionality and its molecular switch. Through structural comparison and, particularly, functional characterization, we revealed that YjjX is a novel ITPase/XTPase responsible for the removal of non-canonical nucleotides from the cell during oxidative stress in Escherichia coli. YhdE, identified as a novel dTTPase, was observed to retard cell growth and form a filamentous phenotype when overexpressed in the cell, suggesting that YhdE is involved in the control of cell growth and division by regulating the cell nucleotide pool for DNA synthesis. In summary, this research has made a substantial ii contribution to the investigation of bacterial phosphorylation and dephophorylation systems that respond to various environmental conditions. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-05-29 11:41:41.832

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "December 2004." Typescript. Vita. Includes bibliographical references (leaves 208-226). Also issued on the Internet.

Proteins are the fundamental machinery that enables the functions of life. It is critical to understand them not just for basic biology, but also to enable medical advances. The field of protein structure prediction is concerned with developing computational techniques to predict protein structure and function from a protein’s amino acid sequence, encoded for directly in DNA, alone. Despite much progress since the first computational models in the late 1960’s, techniques for the prediction of protein structure still cannot reliably produce structures of high enough accuracy to enable desired applications such as rational drug design. Protein structure refinement is the process of modifying a predicted model of a protein to bring it closer to its native state. In this dissertation a protein structure refinement technique, that of potential energy minimization using hybrid molecular mechanics/knowledge based potential energy functions is examined in detail. The generation of the knowledge-based component is critically analyzed, and in the end, a potential that is a modest improvement over the original is presented. This dissertation also examines the task of protein structure comparison. In evaluating various protein structure prediction techniques, it is crucial to be able to compare produced models against known structures to understand how well the technique performs. A novel technique is proposed that allows an in-depth yet intuitive evaluation of the local similarities between protein structures. Based on a graph analysis of pairwise atomic distance similarities, multiple regions of structural similarity can be identified between structures independently of relative orientation. Multidomain structures can be evaluated and this technique can be combined with global measures of similarity such as the global distance test. This method of comparison is expected to have broad applications in rational drug design, the evolutionary study of protein structures, and in the analysis of the protein structure prediction effort.

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