Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

O trabalho realizado como parte do programa de mestrado em física aplicada sub-área biomolecular, teve como objeto de estudo a enzima glicose-6-fosfato isomerase de humanas (PGI-hum). Este trabalho envolveu principalmente três áreas de estudos: biologia molecular, bioquímica e cristalografia. A parte de biologia molecular refere-se a sub-clonagem do gene da PGI-hum a partir de uma biblioteca de cDNA de cérebro de feto humano - capítulo 2 - e a expressão deste gene em bactérias Escherichia coli - capítulo 3. A parte de bioquímica envolve a purificação e caracterização de parâmetros cinéticos da enzima PGI-hum recombinante. Além dos parâmetros cinéticos, foram realizados ensaios de eficiência inibitória para quatro compostos similares ao substrato, cedidos em colaboração com o pesquisador Dr. Laurent Salmon (Lab. De Química Biorgânica e Bioinorgânica de Universidade de Paris-XI). Esta etapa encontra-se descrita nos capítulos 3 e 4. Uma vez definido o protocolo de purificação e confirmada a atividade enzimática para a PGI-hum recombinante, iniciou-se a terceira etapa do projeto: cristalização e determinação da estrutura por difração de raios-X. O primeiro passo, nesta Ultima fase do trabalho, foi determinar as condições de cristalização da PGI-hum - capítulo 5. Depois de obtidos os cristais, foram coletados dois conjuntos de dados de difração de raios-X, sendo o primeiro coletado em uma fonte convencional de raios-X e o segundo com um feixe proveniente de luz sincrotron - capítulo 6. A análise da qualidade e comparação dos conjuntos de dados - capítulo 7 - indicou qual conjunto seria utilizado nas etapas seguintes da determinação da estrutura da PGI-hum. Para resolução da estrutura cristalográfica da PGI-hum foi utilizado o método de substituição molecular com base na estrutura homóloga da PGI de coelho - capítulo 8. O refinamento estrutural da PGI resultou em uma estrutura com 2.1Å de resolução e fatores R e R free satisfatórios - capítulo 9. Uma análise estrutural preliminar é apresentada indicando uma geometria adequada para a proteína e descrevendo as principais características estruturais desta enzima em comparação com a PGI de coelho. / This work is presented as part of the Master degree requirements of the Applied Physics program, Biomolecular Physics area. The purpose of this work is the structural study by of the human glucose-6-phosphate isomerase (PGI-hum). This work has involved mainly three areas: Molecular Biology, Biochemistry and Crystallography. The Molecular Biology work was intended for the cloning of the human open reading frame of PGI-hum from a fetal human brain cDNA library - Chapter 2 - and its expression in Escherichia coli - Chapter 3. The biochemistry work has involved the PGI-hum purification and the determination of its kinetic parameters of the recombinant protein. Inhibitory efficiency measurements where made with four compounds kindly provided by Dr. Laurent Salmon (Laboratoire de Chimie Bioorganique et Bioinorganique - Universite Paris-XI - France). This work is described in Chapters 3 and 4. Once defined the expression and purification protocols and confirmed its enzymatic activity for the recombinant PGI-hum, a third phase was initiated in the project: The crystallization and structure determination by X-ray diffraction. The first step in this last phase of the project was determining the conditions for crystallization of the PGI-hum - Chapter 5. Once obtained the crystals, two data set were collected. One data set was collected \"in house\" X-ray source and a second data set was collected at the Synchrotron beam line (Laboratório Nacional de Luz Sincrotron - LNLS - Campinas) - Chapter 6. The analysis of the quality and the comparison of the two data sets, presented in Chapter 7, indicated that second data set was the best to be used at the following steps. For the resolution of the atomic structure oh the PGL-hum the method of molecular substitution based on the structure of the rabbit homologue enzyme was employed - Chapter 8. The refinement of the PGI-hum structure at 2.1Å resolution and satisfactory R and R free factors is presented in Chapter 9 of this dissertation. A preliminary structural analysis is presented indicating an adequate geometry of the protein and describing the most important structural features of the PGI-hum compared to its homologue, the rabbit PGI.

A Dissulfeto Isomerase Proteína (PDI) é uma chaperona redox essencial responsável pela inserção correta das ligações dissulfeto em proteínas nascentes no retículo endoplasmático. Nesta localização celular, bem como em outras regiões, como na superfície celular, a PDI atua na manutenção da homeostase redox e sinalização. Houve substanciosa evolução no conhecimento sobre a estrutura e funções da PDI, graças a estudos in vitro que utilizam a PDI purificada, quimeras ou seus domínios isolados. Nestas abordagens experimentais, as medidas das atividades redutase e chaperona da PDI são realizadas de forma relativamente simples. Entretanto, medir a atividade isomerase, que é a atividade autêntica da família das PDIs, é tecnicamente bastante complexo. Em células e tecidos, o papel da PDI tem sido descrito com base principalmente em estratégias experimentais de ganho e perda de função. Todavia, ainda há pouca informação na correlação entre os resultados funcionais com a medida das atividades da PDI. Este trabalho compila os principais métodos descritos para medir as quatro atividades da PDI: tiol redutase, tiol oxidase, tiol isomerase e chaperona, com ênfase na descrição de controles e interferentes críticos, como os tampões que contém surfactantes. Ainda, discutir-se-á criticamente os resultados obtidos quando da transposição destes métodos para amostras de homogenatos (celular ou tecidual) / Protein disulfide isomerase is an essential redox chaperone from endoplasmic reticulum, responsible for correct disulfide bond insertion in nascent proteins. At the endoplasmic reticulum and other locations including the cell surface, PDI accounts for redox homeostasis and signaling. Knowledge about PDI structure and function evolved substantially from in vitro studies using purified PDI and chimeras. In these experimental scenarios, PDI reductase and chaperone are readily approachable. However, isomerase activity, the hallmark of PDI family, is significantly complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function experiments. However, there is limited information regarding correlation of these results with PDI activities. In this manuscript, we put together the main methods described for measuring the four PDI activities: thiol reductase, thiol oxidase, thiol isomerase and chaperone, with emphasis on controls and critical interferents, such as detergent-containing buffers. We also discuss the transposition of these methods from purified PDI to cellular or in vivo samples, with critical thoughts about the interpretation of results

Abstract Protein folding of outer membrane and secreted proteins, including receptors, cytokines and antibodies is often linked to disulphide bond formation. Native disulphide bond formation is complex and is usually the rate limiting step in the folding of such proteins. The enzymes which catalyse the slow steps in disulphide bond formation belong to the protein disulphide isomerase (PDI) family. PDI catalyses formation, reduction and isomerization of newly synthesized disulphide bonds. The mechanisms of action of the PDIs are currently poorly understood and this not only inhibits our understanding of the biogenesis of a range of medically important proteins, and hence associated disease states, but also prevents the effective manipulation of the cellular environment by the biotechnology industry for the production of high value therapeutic proteins. Hence, understanding the mechanism of action of these enzymes is vital for a wide range of medically important processes and therapies. In this study the role of a conserved arginine residue in the catalytic activity of PDI was shown. The movement of this residue into and out of the active site locale of PDI was shown to modulate the pKa of the C-terminal active site cysteine of PDI and by that way to allow the enzyme to act efficiently as catalyst both of oxidation and isomerization reactions. The possible role of hydrogen peroxide produced by sulphydryl oxidases during disulphide bond formation was studied in an oxidative protein refolding assay. Analysis showed that hydrogen peroxide can be used productively to make native disulphide bonds in folding proteins with minimal side reactions. In addition, the kinetics of oxidation and reduction of the <b>a</b> domains of PDI and Pdi1p by glutathione was studied in this thesis. The kinetics obtained with stopped-flow and quenched-flow experiments showed the reactions to be more rapid and complex than previously thought. Significant differences exist between the kinetics of PDI and Pdi1p. This implies that the use of yeast systems to predict physiological roles for mammalian PDI family members should be treated cautiously.

Abstract The synthesis of α-hydroxy aldehydes and hydroxymethyl ketones as well as their interconversion to each other are discussed in this thesis. The literature survey of the monograph reviews the synthetic methods for the preparation of 1,2-bifunctionalized hydroxy aldehydes and ketones. The keto-aldehyde isomerisation reaction catalyzed by Triosephosphate isomerase enzyme (TIM) and organic compounds that interact with the TIM are also introduced. In addition, the microwave heating techniques in organic syntheses are reviewed. The practical work consists of two entities: The synthesis of new substrate candidates and transition state analogues for a mutated monomeric TIM. These compounds are model compounds for the catalytic activity and the structural studies of the mutated monomeric TIM. The synthesis of the sulphonyl α-hydroxy ketone-based substrate candidates consists of four successive syntheses. The microwave-activation was utilized in the preparation of a carbon-sulphur bond and the synthesis of hydroxymethyl ketones. The improved synthesis of the terminal α-hydroxy ketone functionality with microwave activation is presented. The formation of charged compounds was utilized to improve the absorption of microwave energy of reaction mixtures. The design and the synthetic work were carried out in accordance to principles of green chemistry. The second part of the practical work is the development of an organocatalytic α-oxybenzoylation reaction of aldehydes with high enantiomeric selectivity. This novel method generated enantiomerically pure α-hydroxy aldehydes in the stable benzoate-protected form from achiral starting materials under mild conditions at the presence of air and moisture.

Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes.

Abstract The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ3-Δ 2-enoyl-CoA isomerase, and (3S)-hydroxyacyl-CoA dehydrogenase activities. The amino-terminal part of perMFE-1 shows sequence similarity to mitochondrial 2-enoyl-CoA hydratases (ECH-1) and Δ3-Δ 2-enoyl-CoA isomerases, and belongs to the hydratase/isomerase superfamily. Family members with known structures are either homotrimers or homohexamers. The purpose of this work was to elucidate the structure-function relationship of the rat perMFE-1 with special reference to the hydratase/isomerase fold. The structural adaptations required for binding of a long chain fatty acyl-CoA were studied with rat ECH-1 via co-crystallization with octanoyl-CoA. The crystal structure revealed that the long chain fatty acyl-CoA is bound in an extended conformation. This is possible because, a flexible loop moves aside and opens a tunnel, which traverses the subunit from the solvent space to the intertrimer space. Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by ECH-1. In the present work the enzymological properties of Glu144Ala and Glu164Ala variants of ECH-1 were studied. The catalytic activity of hydration was reduced about 2000-fold. It was also demonstrated that rat ECH-1 is capable of catalyzing isomerization. The replacement of Glu164 with alanine reduced the isomerase activity 1000-fold, confirming the role of Glu164 in both the hydratase and isomerase reactions. The structural factors favoring the hydratase over the isomerase reaction were addressed studying the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. These mutants had similar enzymatic properties to wild type, thus the catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on interaction with the Gln162 side chain. The perMFE-1 was divided into five functional domains based on amino acid sequence comparisons with the homologous proteins with known structures. Deletion variants of perMFE-1 showed that the folding of an enzymatically active amino-terminal hydratase/isomerase domain requires stabilizing interactions from the two carboxy-terminal domains of perMFE-1. The last carboxy-terminal domain is also required for the folding of the dehydrogenase part of perMFE-1. The dehydrogenase part of perMFE-1 was crystallized.

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.

Currently the mammalian endoplasmic reticulum (ER) is known to contain at least 20 different protein disulphide isomerase (PDI) family members. The oxidoreductases in the PDI family are thought to catalyse the formation and rearrangement of disulphide bonds in newly synthesised proteins. The focus of this work was to characterise two of the PDI family members: PDI and ERp46. In vitro translation reactions of major histocompatibility complex (MHC), β1-integrin (β1-I), haemagglutinin (HA), procollagen α1(III) and preprolaction (pPL) were carried out in untreated or PDI-depleted cells. The depletion of PDI decreased the rate of folding of MHC and β1-I and also prevented the oligomerisation of HA, suggesting a role for PDI in folding these putative substrates. However, when PDI was depleted neither the folding of pPL or HA was affected, implying that they may not be substrates for PDI. To determine the role of ERp46 in the cell, a substrate-trapping approach was used. Here substrates interacting with ERp46 were trapped as mixed disulphides isolated by immunoprecipitation, separated by 2D SDS-PAGE and identified by mass spectrometry. It was demonstrated that ERp46 forms mixed disulphides with at least 23 proteins, including heavily secreted proteins such as laminins, integrins and collagens. In particular, interactions with Ero1, Prx IV, EDEM3 and ERAP2 were found and confirmed by immunoprecipitation of radiolabelled in vitro translated protein. Notably nine of these clients of ERp46 have previously been identified as substrates of ERp57 (Jessop, Watkins et al. 2009). This would support the hypothesis that several different oxidoreductases, working in concert, are required to fold certain substrate proteins. Also, it was confirmed that Prx IV and Ero1 each form a mixed disulphide with PDI. These results highlight the importance of PDI family members in recruiting co-factors to substrates. Additionally, the over-expression of ERp46 led to increased cell survival following DTT treatment, yet after depletion of ERp46, cells were less able to grow, perhaps suggesting a role for ERp46 in maintaining ER redox homeostasis and cell survival. This suggestion was supported by the finding that ERp46 is able to catalyse the reduction of Prx IV in the presence of glutathione. These results suggest that Prx IV provides a novel mechanism for the transfer of disulphide bonds to nascent proteins in the ER via PDI family members such as ERp46 and PDI.

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