Cereals contain naturally occurring biopolymers (for example proteins and starches) that can be used as renewable raw materials in a variety of speciality chemical applications. The separation of protein and starch biopolymers from wheat is well established and relies on a group of proteins called glutens that have a unique network-forming functionality. Oat and other cereals do not naturally contain these gluten proteins and typically rely on chemical-based separation techniques which alter the chemical and physical structures and damage the inherent natural functionality of the biopolymers. This research study investigated the separation of the protein and starch fractions from cereals using the Al-Hakkak Process, a new aqueous process. This process involves adding water and wheat gluten protein to cereals that do not contain gluten. The wheat gluten interacts with the cereal proteins, facilitating the separation of the starch and protein fractions whilst retaining their inherent natural functionality. The aim of this research project was to investigate and optimise the pilot scale separation performance of the Al-Hakkak Process using oat flour. As very little prior research had been carried out, the focus was to characterise the oat starch and protein separation performance and gain an understanding of the mechanisms involved. A variety of techniques were employed. Large scale deformation rheology was used to gain an understanding of the oat-gluten dough rheology and establish the relationship between the rheology and the separation performance. Confocal scanning laser microscopy was used to investigate the structure of the oat-gluten protein network. The molecular interactions between the oat and gluten proteins were studied using gel electrophoresis. The network-forming functionality of the new oat-gluten protein was explored. The influence of various processing parameters on the pilot scale separation performance was investigated and the results compared with other data collected through the study to identify key processing parameters. This research programme has resulted in interesting, encouraging and some unexpected outcomes and these are discussed in detail in the thesis. It was concluded that an insoluble protein network formed in the oat-gluten dough and both kneading and extraction processes were found to contribute to the formation of this. A key conclusion was that the changes that took place in the oat-gluten dough were similar to, but not identical to, the changes that occur in wheat dough. It was proposed that the mechanism for the development of a protein network in oat-gluten dough differed from wheat dough for two main reasons: a) the presence of the oat flour disrupted the normal wheat gluten behaviour, and b) components in the oat flour altered the activity of the gluten proteins. The research identified key processing parameters for the Al-Hakkak Process including kneading time, gluten content, and sodium chloride content of the oat-gluten dough as well as sodium chloride concentration, pH, and temperature of the extract liquor. An important discovery was that the oat and gluten proteins interacted at a molecular level through reducible, covalent, bonding (most likely disulphide linkages) to form the insoluble protein network in the oat-gluten dough. It was concluded that these reducible bonds coupled the individual protein subunits to form new hybrid oat-gluten protein molecules (a combination of oat proteins and gluten proteins). Both insoluble and soluble proteins in the oat and gluten flour were involved in the formation of the insoluble protein network in the oat-gluten dough. This outcome has applications beyond the Al-Hakkak Process, as this new knowledge can be applied to the wider dough processing industry. It was concluded that the wheat gluten was the source of the protein network-forming functionality of the hybrid oat-gluten protein and that the oat proteins had a diluting effect. It was proposed that oat-gluten protein flour from the Al-Hakkak Process could be reused to replace the commercial wheat gluten flour in subsequent production batches. During spray drying of the starch stream, the soluble biopolymers in the extract liquor were found to act as an adhesive and glued individual starch granules together to form spherical agglomerates. Acidification of the extract liquor was found to enhance this agglomeration. It was proposed the acidified starch granules were sticker during spray drying due to the partial acid hydrolysis of the starch granule suface which enhanced the agglomeration.

Despite the fundamental role of experimental protein structure determination, computational methods are of essential importance to bridge the ever growing gap between available protein sequence and structure data. Common structure prediction methods rely on experimental data, which is not available for about half of the known protein families. Recent advancements in amino acid contact prediction have revolutionized the field of protein structure prediction. Contacts can be used to guide template-free structure predictions that do not rely on experimentally solved structures of homologous proteins. Such methods are now able to produce accurate models for a wide range of protein families. We developed PconsC2, an approach that improved existing contact prediction methods by recognizing intra-molecular contact patterns and noise reduction. An inherent problem of contact prediction based on maximum entropy models is that large alignments with over 1000 effective sequences are needed to infer contacts accurately. These are however not available for more than 80% of all protein families that do not have a representative structure in PDB. With PconsC3, we could extend the applicability of contact prediction to families as small as 100 effective sequences by combining global inference methods with machine learning based on local pairwise measures. By introducing PconsFold, a pipeline for contact-based structure prediction, we could show that improvements in contact prediction accuracy translate to more accurate models. Finally, we applied a similar technique to Pfam, a comprehensive database of known protein families. In addition to using a faster folding protocol we employed model quality assessment methods, crucial for estimating the confidence in the accuracy of predicted models. We propose models tobe accurate for 558 families that do not have a representative known structure. Out of those, over 75% have not been reported before. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: In press.</p><p> </p>

No description available.

ATP-dependent chromatin remodellers represent a class of proteins that restructure chromatin through the action of a conserved helicase-like ATPase domain. Remodellers typically have several accessory binding domains alongside the ATPase. These confer target specificity and most commonly recognise histone post-translational modifications. SMARCAD1 is a ubiquitous chromatin remodeller involved with DNA replication and re- pair. It binds directly to PCNA at the site of DNA replication and recruits co-repressor KAP1 in order to silence newly produced chromatin. In contrast to most other chromatin remodellers, SMARCAD1 does not contain several different types of accessory domains. Only two CUE do- mains have been identified in addition to the SMARCAD1 core ATPase domain. CUE domains are a type of helical ubiquitin-binding domain. This thesis presents the findings of an investigation into the structure and function of the SMARCAD1 double CUE domain. The solution NMR structure is presented with results from NMR binding experiments mapped onto the structure. Each CUE domain was found to be an independent helix bundle connected by a dynamic flexible linker. The N-terminal CUE domain, CUE-1, binds ubiquitin and has an adjacent SUMO (a ubiquitin-like protein) binding motif on a protruding extended helix. The C-terminal CUE domain, CUE-2, has a very similar structure to several published CUE domains but does not bind ubiquitin due to a charged substitution at a highly conserved CUE consensus position. The SMARCAD1 double CUE domain binds KAP1 from nuclear extract and is likely to mediate the interaction between SMARCAD1 and KAP1. SMARCAD1 double CUE domain is not involved with PCNA binding.

This thesis describes the development of a novel approach for prediction of the three-dimensional structure of transmembrane regions of membrane proteins directly from amino acid sequence and basic transmembrane region topology. The development rationale employed involved a knowledge-based approach. Based on determined membrane protein structures, 20x20 association matrices were generated to summarise the distance associations between amino acid side chains on different alpha helical transmembrane regions of membrane proteins. Using these association matrices, combined with a knowledge-based scale for propensity for residue orientation in transmembrane segments (kPROT) (Pilpel et al., 1999), the software predicts the optimal orientations and associations of transmembrane regions and generates a 3D structural model of a gi ven membrane protein, based on the amino acid sequence composition of its transmembrane regions. During the development, several structural and biostatistical analyses of determined membrane protein structures were undertaken with the aim of ensuring a consistent and reliable association matrix upon which to base the predictions. Evaluation of the model structures obtained for the protein sequences of a dataset of 17 membrane proteins of detennined structure based on cross-validated leave-one-out testing revealed generally high accuracy of prediction, with over 80% of associations between transmembrane regions being correctly predicted. These results provide a promising basis for future development and refinement of the algorithm, and to this end, work is underway using evolutionary computing approaches. As it stands, the approach gives scope for significant immediate benefit to researchers as a valuable starting point in the prediction of structure for membrane proteins of hitherto unknown structure.

Doctor of Philosophy / Department of Biochemistry / Michal Zolkiewski / ClpB is a bacterial heat-shock protein that disaggregates and reactivates strongly aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two ATP-binding AAA+ modules, a linker coiled-coil domain, and a highly mobile N-terminal domain. It forms ring-shaped hexamers in a nucleotide-dependent manner. The unique aggregation reversing chaperone activity of ClpB involves ATP-dependent translocation of substrates through the central channel in the ClpB ring. The initial events of aggregate recognition and the events preceding the translocation step are poorly understood. In addition to the full-length ClpB95, a truncated isoform ClpB80, that is missing the whole N-terminal domain, is also produced in vivo. Various aspects of the structure and function of ClpB were addressed in this work. The thermodynamic stability of ClpB in its monomeric and oligomeric forms, as well as the nucleotide-induced conformational changes in ClpB were investigated by fluorescence spectroscopy. Equilibrium urea-induced unfolding showed that two structural domains-the small domain of the C-terminal AAA+ module and the coiled-coil domain-were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation or interactions during formation of the ClpB rings. Several locations of Trp-fluorescence probes were also found to respond to nucleotide binding. The biological role of the two naturally-occurring ClpB isoforms was also investigated. We discovered that ClpB achieves optimum chaperone activity by synergistic cooperation of the two isoforms that form hetero-oligomers. We found that ClpB95/ClpB80 hetero-oligomers form preferentially at low protein concentration with higher affinity than homo-oligomers of ClpB95. Moreover, hetero-oligomers bind to aggregated substrates with a similar efficiency as homo-oligomers of ClpB95, do not show enhanced ATPase activity over that of the homo-oligomers, but display a strongly stimulated chaperone activity during the reactivation of aggregated proteins. We propose that extraction of single polypeptides from aggregates and their delivery to the ClpB channel for translocation is the rate-limiting step in aggregate reactivation and that step is supported by the mobility of the N-terminal domain of ClpB. We conclude that the enhancement of the chaperone activity of the hetero-oligomers is linked to an enhancement of mobility of the N-terminal domains.

Proteínas desempenham uma grande variedade de funções biológicas. O conhecimento da estrutura tridimensional proteica pode ajudar no entendimento da função desempenhada. De acordo com a hipótese de Anfisen, a estrutura terciária nativa de uma proteína pode ser determinada a partir da informação contida na sequência primária, o que permitiria que métodos computacionais poderiam ser usados para predizer estruturas terciárias quando a primária estiver disponível. No entanto, ainda não existe uma ferramenta computacional capaz de predizer a estrutura tridimensional para uma grande variedade de proteínas. Desse modo, o problema de Predição de Estruturas de Proteínas (PEP) permanece como um desafio para a Biologia Molecular. A conformação nativa de uma proteína é frequentemente a configuração termodinamicamente mais estável, ou seja, que possui menor energia livre. Assim, PEP pode ser vista como um problema de otimização, onde a estrutura com menor energia livre deve ser encontrada dentre todas as possíveis. Entretanto, este é um problema NP-completo, no qual métodos tradicionais de otimização, em geral, não apresentam um bom desempenho. Algoritmos Genéticos (AGs), devido às suas características, são interessantes para essa classe de problemas. O principal objetivo desse trabalho é verificar se a adição de informação pode ser útil aos AGs aplicados em PEP, valendo-se dede modelos moleculares simplificados. Cada indivíduo do AG representa uma solução que, neste caso, é uma possível conformação que será avaliada por um campo de força. Dessa forma, o indivíduo é codificado por um conjunto de ângulos de torção de cada aminoácido. Para auxiliar no processo de busca, bases de dados compostas de ângulos determinados por cristalografia e RNM são utilizadas. Com o objetivo de guiar o processo de busca e manter a diversidade nos AGs, duas estratégias são aqui testadas: Imigrantes Aleatórios e Imigrantes por Similaridade. A última delas foi criada baseando-se na similaridade da sequência primária. Além disso, é investigado neste trabalho o uso de um campo de força coarse grained, que utiliza os átomos de carbono- para representar a cadeia proteica, para avaliar os indivíduos do AG. / Proteins exhibit an enormous variety of biology functions. The knowledge of tertiary structures can help the understanding of the proteins function. According to Anfisen, the native tertiary structure of a protein can be determined by its primary structure information, what could allow that computational methods could be used to predict the tertiary structure when the primary structure is available. However, there is still not a computational tool to solve the structure prediction problem for a large range of proteins. In this way, Protein Structure Prediction (PSP) has been a challenge to Molecular Biology. The conformation of native protein is usually the thermodynamically most stable configuration, i.e., the one having the lowest free energy. Hence, PSP can be viewed as a problem of optimization, where the structure with the lowest free energy should be found among all possible structures. However, this is an NP-problem, where traditional optimization methods, in general, do not have good performance. Genetic algorithms (GAs), due to their characteristics, are interesting for this class of problems. In recent years, there is a growing interest in using GAs for the protein structure prediction problem. The main objective of this work is to verify the addition of useful information to GAs employed in PSP. Each individual of the GA represents a solution for the optimization problem which is, in this case, a possible conformation that will be evaluated by a force field function. Thus, an individual is encoded by a set of torsion angles of each amino acid. In order to reduce the search space, a database composed of angles, determined by crystallography and NMR, is used. With the aim to guide the final search process and maintain diversity in GAs, two strategies were employed here: Random Immigrants and Similarity-based Immigrants. The last strategy was based on similarity of primary amino acid sequence. Furthermore, in this work, a coarse-grained force field, which uses -carbon to represent the protein backbone was employed to evaluate the individuals of GA.

A produção de soro no Brasil, único tratamento eficaz nos casos de acidentes ofídicos, utiliza equinos que apesar do grande porte, apresentam diminuição da longevidade quando comparado com os cavalos não imunizados. A radiação ionizante tem se mostrado como excelente ferramenta na diminuição da toxicidade de venenos e toxinas isoladas, além de promover a obtenção de melhores imunógenos para a produção de soro. Sabe-se, contudo, que os efeitos da radiação ionizante em materiais protéicos caracterizam-se por diversas alterações químicas, como fragmentação, cross-linking, agregação e oxidação devido aos produtos gerados pela radiólise da água. Porém, a ação da radiação gama em toxinas ainda não está totalmente esclarecida do ponto de vista estrutural e farmacológico, fato que impede a aplicação de tal metodologia no processo de produção do soro. Assim, foi proposto nesse trabalho a caracterização da crotamina, uma das principais proteínas do veneno da espécie Crotalus durissus terrificus, irradiada com raios gama de 60Co. Após o isolamento da toxina por técnicas cromatográficas e a realização de testes para comprovar a obtenção da crotamina pura, a toxina, nos estados nativo e irradiado, foi submetida às análises estruturais de Fluorescência e Dicroísmo Circular. Testes utilizando altas pressões hidrostáticas também foram realizados com o intuito de verificar se as alterações conformacionais causadas pela radiação sofreriam modificações sob altas pressões. Do ponto de vista farmacológico, testes de contração muscular foram realizados com o objetivo de delimitar a ação da crotamina em musculatura lisa, bem como a mudança na ação da toxina frente às alterações estruturais ocasionadas. As análises de Fluorescência e Dicroísmo Circular mostraram que há mudanças na conformação da crotamina quando submetida à radiação gama e que tais alterações possivelmente ocorrem na estrutura terciária e secundária da proteína. O observado nos testes farmacológicos mostraram que a crotamina irradiada foi menos eficaz na diminuição da contração muscular do ducto deferente de ratos em comparação com a crotamina nativa. Além disso, o comportamento da toxina irradiada na contração tônica, modulada por noradrenalina, foi distinto daquele observado para a toxina nativa. / The serum production in Brazil, the only effective treatment in cases of snakebites, uses horses that although large size, have reduced llifespan compared with horses not immunized. Ionizing radiation has been shown as an excellent tool in reducing the toxicity of venoms and toxins isolated, and promote the achievement of better immunogens for serum production, and contributing to the welfare of serum-producing animals. It is known, however, that the effects of ionizing radiation on protein are characterized by various chemical modifications, such as fragmentation, cross-linking due to aggregation and oxidation products generated by water radiolysis. However, the action of gamma radiation on toxins is not yet fully understood structurally and pharmacologically, a fact that prevents the application of this methodology in the serum production process. So we proposed in this paper the characterization of crotamine, an important protein from the venom of Crotalus durissus terrificus species, irradiated with 60Co gamma rays. After isolating the toxin by chromatographic techniques and testing to prove the obtaining of pure crotamine, it was irradiated with gamma rays and subjected to structural analysis, Fluorescence and Circular Dichroism. Using high hydrostatic pressure tests were also conducted in order to verify that the conformational changes caused by radiation suffer modifications under high pressures. From the pharmacological point of view, muscle contraction tests were conducted with the objective of limiting the action of crotamine in smooth muscle as well as the change in the action of toxin caused structural changes to the front. Analysis of Circular Dichroism and Fluorescence showed changes in structural conformation of crotamine when subjected to gamma radiation and that such changes possibly occurring in the secondary and tertiary structure of the protein. The observed in pharmacological tests showed that the irradiated crotamine was less effective in lowering the vas deferens twitch in rats in comparison to native crotamine. In addition, the behavior of irradiated toxin in tonic contraction, modulated by noradrenaline, was different from that observed for the native toxin.

A produção de soro no Brasil, único tratamento eficaz nos casos de acidentes ofídicos, utiliza equinos que apesar do grande porte, apresentam diminuição da longevidade quando comparado com os cavalos não imunizados. A radiação ionizante tem se mostrado como excelente ferramenta na diminuição da toxicidade de venenos e toxinas isoladas, além de promover a obtenção de melhores imunógenos para a produção de soro. Sabe-se, contudo, que os efeitos da radiação ionizante em materiais protéicos caracterizam-se por diversas alterações químicas, como fragmentação, cross-linking, agregação e oxidação devido aos produtos gerados pela radiólise da água. Porém, a ação da radiação gama em toxinas ainda não está totalmente esclarecida do ponto de vista estrutural e farmacológico, fato que impede a aplicação de tal metodologia no processo de produção do soro. Assim, foi proposto nesse trabalho a caracterização da crotamina, uma das principais proteínas do veneno da espécie Crotalus durissus terrificus, irradiada com raios gama de 60Co. Após o isolamento da toxina por técnicas cromatográficas e a realização de testes para comprovar a obtenção da crotamina pura, a toxina, nos estados nativo e irradiado, foi submetida às análises estruturais de Fluorescência e Dicroísmo Circular. Testes utilizando altas pressões hidrostáticas também foram realizados com o intuito de verificar se as alterações conformacionais causadas pela radiação sofreriam modificações sob altas pressões. Do ponto de vista farmacológico, testes de contração muscular foram realizados com o objetivo de delimitar a ação da crotamina em musculatura lisa, bem como a mudança na ação da toxina frente às alterações estruturais ocasionadas. As análises de Fluorescência e Dicroísmo Circular mostraram que há mudanças na conformação da crotamina quando submetida à radiação gama e que tais alterações possivelmente ocorrem na estrutura terciária e secundária da proteína. O observado nos testes farmacológicos mostraram que a crotamina irradiada foi menos eficaz na diminuição da contração muscular do ducto deferente de ratos em comparação com a crotamina nativa. Além disso, o comportamento da toxina irradiada na contração tônica, modulada por noradrenalina, foi distinto daquele observado para a toxina nativa. / The serum production in Brazil, the only effective treatment in cases of snakebites, uses horses that although large size, have reduced llifespan compared with horses not immunized. Ionizing radiation has been shown as an excellent tool in reducing the toxicity of venoms and toxins isolated, and promote the achievement of better immunogens for serum production, and contributing to the welfare of serum-producing animals. It is known, however, that the effects of ionizing radiation on protein are characterized by various chemical modifications, such as fragmentation, cross-linking due to aggregation and oxidation products generated by water radiolysis. However, the action of gamma radiation on toxins is not yet fully understood structurally and pharmacologically, a fact that prevents the application of this methodology in the serum production process. So we proposed in this paper the characterization of crotamine, an important protein from the venom of Crotalus durissus terrificus species, irradiated with 60Co gamma rays. After isolating the toxin by chromatographic techniques and testing to prove the obtaining of pure crotamine, it was irradiated with gamma rays and subjected to structural analysis, Fluorescence and Circular Dichroism. Using high hydrostatic pressure tests were also conducted in order to verify that the conformational changes caused by radiation suffer modifications under high pressures. From the pharmacological point of view, muscle contraction tests were conducted with the objective of limiting the action of crotamine in smooth muscle as well as the change in the action of toxin caused structural changes to the front. Analysis of Circular Dichroism and Fluorescence showed changes in structural conformation of crotamine when subjected to gamma radiation and that such changes possibly occurring in the secondary and tertiary structure of the protein. The observed in pharmacological tests showed that the irradiated crotamine was less effective in lowering the vas deferens twitch in rats in comparison to native crotamine. In addition, the behavior of irradiated toxin in tonic contraction, modulated by noradrenaline, was different from that observed for the native toxin.

A contração muscular esquelética é regulada pelo complexo troponina/tropomiosina de maneira dependente de Ca2+. O complexo troponina consiste de três subunidades: a troponina C (TnC), a troponina I (TnI) e a troponina T (TnT). A troponina C é a subunidade que liga Ca2+, a TnI é a subunidade inibitória e a TnT liga-se fortemente à tropomiosina. A TnI e a TnT são altamente insolúveis a baixas forças iônicas, a não ser que estejam complexadas com a TnC. O complexo troponina pode ser reconstituído \"in vitro\" a partir das subunidades isoladas simplesmente misturando-se as subunidades em razões equimolares em uréia, que depois é removida através de diálise. Na primeira parte deste trabalho um vetor para a co-expressão da TnC, TnI e TnT em E.coli foi construído. Utilizando este vetor nós produzimos um complexo troponina funcional montado no citoplasma de E.coli. A presença da TnT é requerida para regulação dependente de Ca2+ da contração muscular esquelética. O papel da TnT em conferir sensibilidade ao Ca2+ à atividade ATPásica da acto-miosina foi analisado. Mutantes de deleção da TnT foram construídos através de mutação sítio-dirigida e expressos em E.coli. Complexos troponina contendo os mutantes de TnT e/ou mutantes de TnI foram reconstituídos e analisados em ensaios de ligação ao filamento fino e ensaios de atividade ATPásica. Baseado nestes resultados a TnT foi subdividida em três domínios: o domínio ativatório (aminoácidos 157-216), o domínio inibitório (aminoácidos 157-216) e o domínio de ancoragem do dímero TnC/TnI (aminoácidos 216-263). Nós demonstramos que o dímero TnC/TnI está ancorado ao filamento fino através da interação entre a região amino-terminal da TnI e da região carbóxi-terminal da TnT (aminoácidos 216-263). Um modelo para o papel da TnT na regulação da contração muscular dependente de Ca2+ é proposto. / The contraction of skeletal muscle is regulated by troponin and tropomyosin in a Ca2+ dependent manner. The troponin complex consists of three subunits: troponin C (TnC), troponin I (TnI) and troponin T (TnT). Troponin C is the Ca2+ binding subunit, TnI is the inhibitory subunit and TnT binds tightly to tropomyosin. TnI and TnT are highly insoluble proteins at low ionic strengths, unless they are complexed with TnC. The troponin complex can be reconstituted \"in vitro\" from the isolated subunits simply by mixing the subunits at equimolar ratios in urea, which is then removed by dialysis. In the first part of this work a vector for the co-expression of TnC, TnI and TnT in E.coli was constructed. Using this vector we were able to produce a functional troponin complex assembled \"in vivo\" in the E.coli cytoplasm The presence of TnT is required for the Ca2+ dependente regulation of the skeletal muscle contraction. The role of TnT in conferring full Ca2+ sensitivity to the ATPase activity of acto-myosin was analyzed. Deletion mutants of TnT were constructed by site-directed mutagenesis and expressed in E.coli. Troponin complexes containing the TnT deletion mutants and/or TnI deletion mutants, were reconstituted and analyzed in thin filament binding assays and in ATPase activity assays. Based on these studies, TnT was subdivided into three domains: the activation domain (comprised of aminoacids 1-157), the inhibitory domain (comprised of amino acids 157-216) and the TnC/TnI dimer anchoring domain (aminoacids 216-263). We demonstrated that the TnC/TnI is anchored to the thin filament through interaction between the amino-terminal domain of TnI and the region comprised of aminoacids 216-263 of TnT. A model for the role of TnT in the Ca2+ dependent regulation of muscle contraction is proposed.