An experimental study on randomly generated protein sequences can provide important insights into the origin and mechanism of secondary structure formation and protein folding. In this study we bring biophysical characterization of five protein sequences selected from the in silico generated library of random chains. The sequences were selected on the basis of bioinformatic analysis in order to find the candidates with the maximum potential to possess secondary structure. This study shows that the random polypeptide sequences form stable secondary structures and in some show the signs of tertiary structure, such as hydrophobic core formation and distinctive oligomerization pattern. While the work presented in this thesis is work in progress on a larger study, the data already demonstrate that unevolved protein sequence space provides a lot of potential for secondary and tertiary structure formation that awaits its characterization. Powered by TCPDF (www.tcpdf.org)

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.

Traditional computational protein design (CPD) calculations model sequence perturbations and evaluate their stabilities using a single fixed protein backbone template in an approach referred to as single‐state design (SSD). However, certain design objectives require the explicit consideration of multiple conformational states. Cases where a multistate framework may be advantageous over the single‐state approach include the computer aided discovery of new enzyme substrates, the prediction of protein stabilities, and the design of protein dynamics. These design objectives can be tackled using multistate design (MSD). However, it is often the case that a design objective requires the consideration of a protein state having no available structure information. For such circumstances the multistate framework cannot be applied. In this thesis I present the development of two template and ensemble preparation methodologies and their application to three projects. The purpose of which is to demonstrate the necessary ensemble modeling strategies to overcome limitations in available structure information. Particular emphasis is placed on the ability to recapitulate experimental data to guide modelling of the design space. Specifically, the use of MSD allowed for the accurate prediction of a methyltransferase recognition motif and new substrates, the prediction of mutant sequence stabilities with quantitative accuracy, and the design of dynamics into the rigid Gβ1 scaffold producing a set of dynamic variants whose tryptophan residue exchanges between two conformations on the millisecond timescale. Implementation of both the ensemble, coordinate perturbation followed by energy minimization (PertMin), and template, rotamer optimization followed by energy minimization (ROM), generation protocols developed here allow for exploration and manipulation of the structure space enabling the success of these applications.

RNA silencing is a cellular mechanism of post-transcriptional gene regulation which is highly conserved among the plant and animal kingdoms of life, and plays a critical part of developmental biology, maintenance of homeostasis, and host-pathogen interactions. The pathway is engaged by small double-stranded (ds)RNA molecules (small RNAs), which effect sequence specific gene silencing by targeting complementary RNA sequences. There are several classes of small RNAs which engage the pathway. MicroRNAs (miRNAs) are expressed in the genome as endogenous regulators of gene expression. Short-interfering RNAs (siRNAs) are usually from exogenous sources such as viral-derived short-interfering RNAs, or synthetic siRNAs which are applied to cells or organisms to inhibit expression of specific genes. The p19 protein is a viral suppressor of RNA silencing (VSRS) endogenous to tombusviruses, which binds small RNA duplexes of any sequence with extremely high affinity. Because of its unique binding properties, recombinant p19 proteins are an excellent platform for tool development surrounding the RNA silencing pathway and are used extensively in novel applications for modulating the activity of small RNAs in living systems and for detecting small RNAs in biological samples. Herein we present work that has increased the breadth of p19’s utility as a biotechnology tool in three distinct realms. First, we present a chemical biology approach which combines p19 and small molecules for potent inhibition of the RNA silencing pathway in human cells. Secondly, we present the development of a novel fusion protein between p19 and a cell penetrating peptide (CPP), which functions as an siRNA delivery agent to allow gene knockdown in human cells. Thirdly, we have improved the utility of p19 for detecting and sequestering human miRNAs through rationally designing the binding surface; we describe mutations which dramatically enhance p19's affinity for human miRNA-122. The work presented here adds to the growing repertoire of engineered RNA binding proteins (RBPs) as tools for studying small RNA molecules and modulating their activity for applications in human therapeutics.

Orientadores: Glaucia Maria Pastore, Fábio Márcio Squina / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T10:16:22Z (GMT). No. of bitstreams: 1 Silva_JunioCota_D.pdf: 6755717 bytes, checksum: 8ce3ba1fb6b01179b70462bf26349499 (MD5) Previous issue date: 2013 / Resumo: Atualmente há uma crescente demanda para o desenvolvimento de combustíveis não-fósseis alternativos. Assim, como a biomassa lignocelulósica é uma das fontes de energia mais abundantes na natureza, pode ser estabelecida uma economia verde e sustentável, com o objetivo de processar a grande quantidade de energia estocada nessas matérias-primas. O etanol de cana-de-açúcar é uma das melhores opções em biocombustíveis e sua produção pode mais que dobrar, se os açúcares constituintes da parede celular vegetal forem utililizados. No entanto, o alto custo de produção das enzimas para hidrolisar e processar os materiais lignocelulósicos é um fator altamente limitante para o uso de tecnologias verdes. Este trabalho se propôs a avaliar novos biocatalisadores e construir uma enzima quimérica na tentativa de obter glicosidases com melhor desempenho que as já relatadas. Enzimas despolimerizadoras de ß-1,3-glucanos têm consideráveis aplicações biotecnológicas, incluindo produção de biocombustíveis, insumos químicos e farmacêuticos. No segundo capítulo, mostramos a caracterização funcional e a estrutura de baixa resolução da laminarase hipertermofílica de Thermotoga petrophila (TpLam), além de seu modo de operação por eletroforese capilar de zona, mostrando que ela cliva especificamente ligações ß-1,3-glucosídicas internas. O dicroismo circular (CD) UV-distante demonstrou que TpLam é formada principalmente por elementos estruturais do tipo beta, e a estrutura secundária é preservada após incubação por 16 horas a 90 º C. A forma determinada pelo pequeno espalhamento de raios-X a baixo ângulo revelou uma arquitetura de multi-domínio da enzima, com um arranjo de envelope em forma de V, no qual os dois módulos de ligação de carboidrato estão ligados ao domínio catalítico. A engenharia de enzimas multifuncionais pode melhorar coquetéis enzimáticos para tecnologias emergentes de biocombustíveis. Dinâmica molecular através de modelos baseados em estrutura (SB) é uma ferramenta eficaz para avaliar a disposição tridimensional das enzimas quiméricas, bem como para inferir a viabilidade funcional antes da validação experimental. No terceiro capítulo, descrevemos a montagem computacional de uma quimera bifuncional xilanase-liquenase (XylLich), usando os genes xynA e bglS de Bacillus subtilis. As análises in silico da área de superfície acessível ao solvente (SAS) e da raiz quadrada média das flutuações (RMSF) previram uma quimera completamente funcional, ou seja, uma enzima cujo substrato tem acesso ao seu sítio catalítico com pequenas flutuações e variações ao longo das cadeias polipeptídicas. A quimera preservou as características bioquímicas das enzimas parentais, com exceção de uma pequena variação na temperatura de operação e na eficiência catalítica (kcat / Km). Também foi verificado ausência de mudanças significativas no modo de operação catalítico. Além disso, a produção de enzimas quiméricas pode ser mais rentável do que a produção de uma única enzima separadamente, comparando-se o rendimento da produção de proteína recombinante e a atividade hidrolítica da enzima quimérica com as enzimas parentais. ß-Glicosidases (BGLs) são enzimas muito úteis e com grande potencial para serem empregadas em diversos processos industriais. Entretanto, algumas características são essenciais para tornar viáveis as aplicações, como por exemplo estabilidade à temperatura e ao pH, bem como baixa inibição por íons e outros compostos químicos. Assim, no quarto capítulo buscamos estudar três BGLs dos organismos extremófilos Pyrococcus furiosus e Thermotoga petrophila. Os genes PfBgl1, TpBgl1 and TpBgl3 foram clonados no vetor pET28a e as proteínas expressadas em Escherichia coli e posteriormente purificadas em duas etapas cromatográficas. As enzimas purificadas foram avaliadas quanto ao pH e temperatura de atividade, sendo que as BGLs da família GH1 (PfBgl1 e TpBgl1) apresentaram faixas mais largas de pH e temperatura de operação do que a família GH3 (TpBgl3). As BGLs mostraram grande estabilidade ao pH e o maior tempo de meia-vida (a 99 ° C) foi verificado no pH 6, e além disso, não foram significativamente afetadas pela presença de EDTA ou de íons, exceto a TpBgl1 que foi inibida por Hg2+ e Fe2+. As atividades específicas para um conjunto de diferentes substratos sugeriram que TpBgl3 é mais específica que as BGLs GH1. O kcat e kcat / Km em 4-nitrofenol-ß-D-glicopiranosídeo (pNPG) indicam que TpBgl3 é a mais eficiente para hidrólise do substrato, embora seja a enzima que foi inibida com a menor concentração de glicose (30.1 mM). Além disso, as BGLs foram analisadas quanto à influência de seis monossacarídeos na catálise, e demonstraram serem fracamente inibidas pela maioria dos açúcares testados. Os ensaios de CD UV-distante revelaram que a estrutura secundária das BGLs não é afetada pelas variações de pH, e os estudos de desnaturação térmica evidenciaram que as BGLs são proteínas hipertermofílicas / Abstract: There is an increasing demand for the development of alternative non-fossil fuels. Thus, since the lignocellulosic biomass is the most abundant source in nature, it may be settled a green and sustainable economy, aiming to process the great amount of energy stocked in these raw materials. The ethanol from sugarcane is one of the best options concerning biofuels and its productivity could be raised more than double if the use of sugars constituents of plant cell wall is considered. However the high production cost of the enzymes to hydrolyze and process lignocellulose is a great limiting factor for green technologies. In this way, this work proposed to evaluate new enzymes and engineer a chimeric enzyme in the attempt to prospect glycosyl hydrolases with better performance than those reported up to date. 1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including the production of biofuels, feedstock-chemicals and pharmaceuticals. In the first chapter we showed the comprehensive functional characterization and low-resolution structure of hyperthermophilic laminarase from Thermotoga petrophila (TpLam), besides its mode of operation through capillary zone electrophoresis, which specifically cleaves internal ß-1,3-glucosidic bonds. Far-UV circular dichroism demonstrated that LamA is formed mainly by beta structural elements, and the secondary structure is maintained after incubation up to 16 hours at 90ºC. The structure determined by small angle X-ray scattering revealed a multi-domain structural architecture of the enzyme with a V-shape envelope arrangement of the two carbohydrate binding modules in relation to the catalytic domain. Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional disposal of chimeric enzymes as well as for inferring the functional practicability before experimental validation. In the second chapter we describe the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average surface accessible area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, i.e. the substrate has access to the catalytic pocket with minor fluctuations and variations along the polypeptide chains. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes. ß-Glucosidases (BGLs) are very useful enzymes with a great potential to be employed in several industrial processes. However, some features are required to become viable the enzyme applications, such as temperature and pH stability as well, low ions and chemicals inhibition. Thus this work aimed to study three BGLs from the extremophiles organisms Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1, TpBgl1 and TpBgl3 were cloned into pET28a vector and the proteins were expressed in Escherichia coli and further purified in two chromatographic steps. The purified enzymes were evaluated for pH and temperature of activity, which showed that BGLs from the glycosyl hydrolases family 1 (PfBgl1, TpBgl1) presented a wider range of pH and temperature operation than BGL from family 3 (TpBgl3). The BGLs showed great stability to a range of pH (4-10) and the highest time of half-life (at 99 °C) was at pH 6, besides they were not significantly affected by the presence of EDTA or ions, except TpBgl1 that was inhibited by Hg2+ and Fe2+. The specific activities in a set of different substrates suggested that TpBgl3 is more specific than GH1 BGLs. The kcat and kcat/Km in pNPG indicate that TpBgl3 is the most efficient among BGLs characterized herein, although this enzyme is inhibited with the lowest glucose concentration (30.1 mM). Furthermore, the BGLs were assayed for influence of six monosaccharides in catalysis, which the results suggested a weak inhibition by the most of those carbohydrates tested. The CD experiments revealed that the secondary structure of BGLs is not affected by the pH variations and the denaturation studies evidenced that the BGLs are indeed hyperthermophilic / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

Novel biocatalysts are highly demanded in the white biotechnology. Hence, the development of highly stable and enantioselective biocatalysts with novel functionalities is an ongoing research topic. Here, an osmium ligating single-site ArM was created based on the biotinstreptavidin technology for the dihydroxylation of olefins. For the creation of the artificial catalytic metal center in the streptavidin (SAV) cavity, efficient osmium tetroxide (OsO4) chelating biotin-ligands were created. The unspecific metal binding of the host scaffold was diminished through genetical and chemical modification of the host protein. The created single-site OsO4 chelating ArM was successfully applied in the asymmetric cyclopropanation, revealing a stable and tunable catalytic hybrid system for application. The structural analysis of protein-ligand complexes is essential for the advanced rational design and engineering of artificial metalloenzymes. In previous studies, a SAV-dirhodium ArM was created and successfully applied in the asymmetric cyclopropanation reaction. To improve the selectivity of the SAV-dirhodium complex, the structural location of the organometallic complex in the SAV cavity was targeted and small-angle x-ray scattering (SAXS) was used to obtain the structural information. The SAXS analysis revealed valuable information of the molecular state of the complexes; hence, the method proved to be useful for the structural analysis of protein-ligand interactions. The discovery of novel enzymes from nature is still the major source for improved biocatalysts. One of the most important enzymes used in the molecular biology are DNA polymerases in PCR reactions. The halothermophilic brine-pool 3 polymerase (BR3 Pol) from the Atlantis II Red Sea brine pool showed optimal activities at 55 °C and salt concentrations up to 0.5 M NaCl, and was stable at temperatures above 95 °C. The comparison with the hyperthermophilic KOD polymerase revealed the haloadaptation of BR3 Pol due to an increased negative electrostatic surface charge and an overall higher structural flexibility. Engineered chimeric KOD polymerases with swapped single BR3 Pol domains revealed increased salt tolerance in the PCR, showing increased structural flexibility and a local negative surface charge. The understanding of the BR3 Pol haloadaptation might enable the development of a DNA polymerase tailored for specific PCR reactions with increased salt concentrations.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21836号 / 農博第2349号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5208(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 三芳 秀人, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

AG to A transition at position -76 in the promoter region of the apoAI gene was previously identified, and the A-76 has been shown to be associated with high apoAI levels. The functional significance of the point mutation was assessed by analysing the DNA-protein binding and promoter activities of the different alleles. This data would suggest that the point mutation alters the function of the apoAI promoter as gel retention assays revealed that the G fragment (-140 to +10) formed an extra DNA-protein complex compared to the A fragment (-140 to +10). Concurrent with the altered DNA-protein interaction between the G and the A fragments, the transcriptional activities of the apoAI gene were found to also be altered. CAT assays have indicated a 1.91 fold increase in promoter activity of the A fragment as compared to the G fragment (-256 to +397). The difference in promoter activity was, however, highly dependent on the particular fragment used, as no difference was observed between the alleles when a fragment {-256 to +68) was used. In this study elements were identified in the region +68 to +397 that causes a reduction in the promoter activity of the G allele by 3.6 fold, whilst reducing the A allele activity by 2 fold. This data would suggest that the point mutation functionally alters the apoAI promoter activity via its interaction with other sequences especially in the region +68 to +397.

No description available.