Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). "July 2006." Includes bibliographical references (leaves 69-72). Also available via the World Wide Web.

Thesis (Ph. D.)--University of Oregon, 2000. / Includes vita and abstract. Includes bibliographical references (p. 199-211). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (p. 166-172).

<p>Medical advances often come as a result of understanding the underlying mechanisms of life. Life, in this sense, happens at various scales. A very complex and interesting one is the molecular scale. Understanding life’s mechanistic details at this level will provide the most promising therapies to modern ailments. Because of structure and function’s close relationship, knowledge of macromolecular structure provides invaluable insight into molecular mechanism.</p><p>A major tool used to get structural information at the molecular scale is X-ray crystallography. Such experiments result in an electron density map from which a model is built. Building such a model is a difficult task, especially at low resolu- tion where detailed features in the electron density deteriorate making it difficult to interpret. However, many advances in the field have greatly eased the model build- ing task, in fact, at high resolutions it has become automated. However, human inspection is still required to get a correct solution.</p><p>The largest boon to model building has been the application of structural knowl- edge. A prominent example is bond and dihedral angles. We often know what is absolutely not allowed and often convince ourselves we know everything that is al- lowed. This work focuses on the fuzzy border between allowed and disallowed. The hypothesis is that rare structural conformations exist but one needs to take great care in modeling them.</p><p>This work has two major components – rotamers (protein sidechain conformation)</p><p>and Hoogsteen base pairing in DNA. I first describe methods used to gain empirical knowledge about rotamers and how that knowledge is used in model validation. Part of this knowledge is rotamer-dependent bond angle deviations. I describe how the observation and quantitation of these deviations is used in a novel set of restraints in protein structure refinement. To provide structural context to rare rotamers, I describe where and why some occur.</p><p>My DNA work has focused on Hoogsteen base pairing. I describe a collaborative survey of existing Hoogsteen base pairs in the PDB. Lessons learned during the survey led to the other DNA topic, the detection and correction of mismodeled purines. I identified Hoogsteens in the PDB mismodeled as Watson-Crick base pairs. This work underscores that Hoogsteens are extremely rare but nonetheless do occur.</p><p>The fuzzy borderland between allowed and disallowed is a strange place filled with the most interesting structural features. My work here has focused on this area, bringing into view many rare conformations. Going forward we need to ensure that conformational frequency is taken into account during model building, refinement, and validation.</p> / Dissertation

abstract: Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging. / Dissertation/Thesis / Ph.D. Chemistry 2012

TAX1BP1 is a pleiotropic multi-domain protein involved in many important biological processes such as signal transduction, cell growth and apoptosis, transcriptional coactivation, membrane trafficking, neurotransmission and autophagy. The N-terminus of TAX1BP1 contains a SKICH domain implicated in autophagy. SKICH domains are also present in four other proteins including NDP52, CALCOCO1, SKIP and PIPP. The SKICH domains of SKIP and PIPP mediate plasma membrane localization. The functions of the SKICH domains of NDP52 and CALCOCO1 are not known. We solved the crystal structure of the TAX1BP1 SKICH domain, which has an Ig-like fold similar to the NDP52 SKICH domain. Extensive pairwise and clustered aromatic π-stacking interactions are present in the TAX1BP1 SKICH domain. The aromatic residues mediating these interactions can be classified into four groups with varying degrees of conservation among different protein families. The interactions mediated by highly conserved residues are found in the interior and one outward face of the Ig-like β-barrel, representing common structural features of the SKICH domains. Three TAX1BP1-specific pairwise interactions locate in the loop regions, each augmented by a proline-aromatic interaction. The three proline-aromatic clusters are linked together by more generic hydrophobic interactions, forming a unique hydrophobic surface at one end of the TAX1BP1 SKICH domain. The structures and homologous models of SKICH domains from different proteins reveal substantial differences in electrostatic surface properties of the domains. Together with existing biochemical data, results from the structural study suggest that an intact SKICH domain is required for the autophagy function of TAX1BP1.

Adviser: Brian W. Matthews. xii, 211 p. ; ill. (some col.) A print copy of this title is available through the UO Libraries under the call number: SCIENCE QP609.G3 J84 2000 / An atomic-level description of the presumed catalytic action of β-galactosidase is described. This large enzyme, from E. coli , carries out two reactions which allow the bacterium to live on the disaccharide lactose. First, it breaks down lactose to the two monosaccharides galactose and glucose. Second, it converts lactose into another disaccharide, allolactose, which is the inducer for the lac operon, and thus is the signal to the bacterium to produce more β-galactosidase. The work is based on high resolution x-ray crystallography and enzyme kinetics. A crystal form of β-galactosidase was isolated that permits data collection up to 1.5 à resolution. Using this crystal form, the structures of several ligands bound to the enzyme were determined. These ligands were chosen to mimic various points in the reaction: binding of substrate, covalent intermediates, transition states, and products. Together these complexes suggest a reaction coordinate for β-galactosidase which clarifies and enhances previous ideas about the reaction mechanism. The reaction includes a conformational change triggered by the progression of the substrate towards the transition state. Additional investigation suggests that this conformational change is involved in determining whether the enzyme carries out its hydrolysis or isomerization reaction. Considerations of the structure in the context of other related enzymes suggest an evolutionary path for β-galactosidase. It is suggested that a progenitor enzyme which catalyzed the hydrolysis of long polysaccharide substrates recruited additional domains which permit β-galactosidase to act on smaller substrates and produce the inducer, allolactose. This dissertation includes both my previously published and co-authored material.

Carrageenans are sulfated polysaccharides found in the cell walls of red algae with 20 – 30 % of the dry weight coming from sulfate esters. The understanding of how heterotrophic bacteria desulfate and depolymerize carrageenan has become a rather arduous endeavor as there are 15 different classes of carrageenan distinguished by the degree of sulfation and the presence or absence of a unique galactose derivative, the 3,6-anhydro-D-galactose. The depolymerization of carrageenan requires the removal of the sulfate substituents, a role fulfilled by sulfatases, which hydrolyze sulfate esters playing a key role in the regulation of sulfation states that determine the function of sulfated biomolecules. Through structural, mechanistic, and sequence-based studies a highly conserved sulfate-binding motif has been identified among sulfatases; however, the molecular determinants for substrate specificity remain largely speculative. Additionally, the largest sulfatase family S1, requires a unique catalytic residue resulting from a post-translationally modified cysteine in order to be functional thus making them difficult to study in vitro. Using a strain of Pseudoalteromonas sp. PS47 isolated in the Boraston Lab I show that the depolymerization of carrageenan is dependent on the degree of sulfation and that recognition of the leaving group is the driving force behind S1 specificity. With little information on the recognition of sulfated biomolecules, the X-ray crystal structures of the three sulfatases from PS47; PsS1_19A, PsS1_19B, and PsS1_NC in complex with their biological substrates provides a deeper understanding of how carbohydrate specific sulfatases recognize their cognate substrate and how this recognition of the leaving group can be extended to other S1 sulfatase families. Furthermore, I show that an exo-acting glycoside hydrolase (PsGH42) requires desulfation of carrageenan oligosaccharides before it can hydrolyze the β-glycosidic linkage, a new specificity of family 42. This research demonstrates how carrageenan depolymerization is entirely dependent on the functionality and specificity of the sulfatases found within the carrageenan utilization locus. / Graduate / 2019-12-07

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-29Bitstream added on 2014-06-13T19:28:55Z : No. of bitstreams: 1 barros_ac_me_botib_parcial.pdf: 177767 bytes, checksum: 6e03eeb7f2b4d288bf363856776ce73f (MD5) Bitstreams deleted on 2015-06-03T11:42:50Z: barros_ac_me_botib_parcial.pdf,. Added 1 bitstream(s) on 2015-06-03T11:44:13Z : No. of bitstreams: 1 000691671_20150729.pdf: 177273 bytes, checksum: 1165399c11c2160349be3e18d5484858 (MD5) Bitstreams deleted on 2015-08-03T12:21:12Z: 000691671_20150729.pdf,. Added 1 bitstream(s) on 2015-08-03T12:22:25Z : No. of bitstreams: 1 000691671.pdf: 1177243 bytes, checksum: 866f3caf560a4a70badf707c949ccf65 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A estabilidade e integridade do material genético são fundamentais para manutenção e continuação da vida. O genoma humano é composto por três bilhões de pares de bases, os quais codificam entre 30.000 – 40.000 genes, e é constantemente atacado por metabólitos reativos endógenos, drogas terapêuticas e uma infinidade de agentes mutagênicos ambientais que afetam sua integridade. Assim, é evidente que a estabilidade do genoma deve estar sob vigilância contínua. Isso é conseguido através de mecanismos de reparo de DNA, que se desenvolveram para remover ou tolerar lesões e erros no DNA. Dentre os mecanismos de reparo de DNA presentes nos organismos, eles podem ser divididos em: i) reparo por excisão de bases (BER), ii) excisão de nucleotídeos (NER), iii) mal-pareamento de bases (MMR) e iv) reparo de DNA por junção de terminais não homólogos (NHEJ). Para o que esses mecanismos funcionem de maneira adequada, é evidente a importância da interação entre proteínas responsáveis pela função de reparo, bem como é essencial a regulação da importação nuclear para localização correta das proteínas responsáveis pelos mecanismos mencionados. Dentre os mecanismos responsáveis pela regulação da importação nuclear, a via clássica constituída pelo heterodímero Importina-α/β é um dos principais mecanismos de deslocamento. Algumas proteínas de reparo parecem interagir somente com algumas isoformas da Importina-α (Imp), indicando uma regulação adicional do processo de reparo, porém, pouco se sabe a respeito do reconhecimento das sequências de localização nuclear (NLS) dessas proteínas. Esse trabalho trata especificamente do estudo do complexo estrutural de complexos de Imp com peptídeos NLSs de proteínas relacionadas ao reparo de DNA utilizando... / The stability and integrity of the genetic material are essential for the maintenance and continuation of life. The human genome, comprising three billion base pairs coding for 30.000 – 40.000 genes, is constantly attacked by endogenous reactive metabolites, therapeutic drugs and a plethora of environmental mutagens that impact its integrity. Thus it is obvious that the stability of the genome must be under continuous surveillance. This is accomplished by DNA repair mechanisms, which were developed to remove or tolerate injuries and mistakes in DNA. In the DNA repair mechanisms available in the organisms, they can be divided in: i) base excision repair (BER), ii) nucleotide excision repair (NER), iii) mismatch repair (MMR) and iv) DNA repair by non-homologous end joining (NHEJ). To an adequate operation of these mechanisms, it is evident the importance of the interaction between proteins responsible for the function of DNA repair, as well as the regulation for nuclear import is essential for the correct localization of the proteins responsible for the mentioned mechanisms. In the group of the mechanisms responsible for nuclear import regulation, the classical pathway composed by Importin-α/β heterodimer is one of the major mechanisms of transport. Some DNA repair proteins seem to interact only with certain isoforms of Importin-α (Imp), indicating an additional regulation of the repair process. However, little information is known about the recognition of nuclear localization sequences (NLS) of these proteins. This work concerns specifically the structural studies of complexes with Imp and NLSs peptides from proteins related to DNA repair using crystallographic techniques. The expression and purification of Impα from Mus musculus N-terminally truncated were performed as well as the co-crystalization of... (Complete abstract click electronic access below)