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Structural properties of phosphoglucose isomerases isolated from a variety of species have been compared by peptide fingerprinting, predicted amino acid sequence homologies and by denaturation and renaturation studies. The enzymes are more readily denatured in guanidinium chloride than in urea, and the isomerase isolated from yeast is more stable toward acid pH than the rabbit muscle enzyme. The rates of guanidinium chloride-induced denaturation are markedly increased by ionic strength and decreased by substrates, competitive inhibitors or glycerol. The enzyme can be renatured, but only in the presence of glycerol. The renaturation process is dependent on protein concentration and temperature and provides a method for the formation of mixed species heterodimers.

The thermal denaturation of proteins has been extensively studied using several methods including differential scanning calorimetry (DSC). A custom-built optical system was used to study thermal effects on protein as an alternative method to DSC measurements. It was used to investigate the thermal stability of bovine serum albumin (BSA) with a focus on comparisons with published DSC data. In the first study, the effect of protein concentration on the thermal denaturation (Td) of BSA was determined and validated using published DSC data for bovine serum albumin (BSA). The optical rotation (OR) and transmitted light (TL) signals indicating protein conformational changes and gel formation, respectively, were collected during the heating of BSA solutions at ~6��C/min from room temperature to ~85��C. The experiments were performed on 1, 2.5 and 5% (w/v) BSA in 0.01 M phosphate buffer at pH 7 and ionic strength (IS) 0.08. BSA���s Td values obtained from this investigation were consistent with published values and had low experimental variability (CV<2.5%). In agreement with some but not all published data, increasing BSA concentration did not affect its thermal stability. Protein gel formation, however, increased with protein concentration. In the second study, changes in the OR and TL signal of BSA in 0.01 M phosphate buffer at pH 6.1, 7 and 7.9 with IS maintained at 0.04, 0.08 and 0.16 were recorded during the heating of BSA solutions at ~6��C/min from room temperature to ~85��C. BSA showed a maximum and minimum thermostability at pH 7 and 7.9, respectively, consistent with published values determined by DSC. BSA formed opaque gel at pH 6.1 approaching the BSA���s pI values. Increasing IS level did not have a significant effect on BSA���s Td value but promoted gel formation. In the third study, the optical method was applied to investigate the heat stability of BSA as affected by low concentrations of sucrose, trehalose or sorbitol. BSA solutions (2.5% w/v) in the presence of 0 5% sucrose, trehalose and sorbitol were heated at ~6��C/min from ambient temperature to ~85��C. In contrast with published work on the thermal stability of BSA in the presence of higher sugar concentrations, this study showed that increasing sugar concentration did not enhance the thermal stability of this protein. Also, the ability to promote protein stability among sucrose, trehalose and sorbitol were not significantly different. The significance of these studies is that they demonstrate that the custom-built optical methods here developed can be used to study heat-induced protein denaturation and the effect of environmental conditions. Future studies will examine other proteins such as ��-lactoglobulin or ��-lacactalbumin. A further advantage of optical systems is their ability to conduct real-time measurements which could be used for food processing control. / Graduation date: 2007

The aim of this thesis is to study the energy fluctuations (leading to thermal fluctuations) during thermal and enzymatic denaturation of biological molecules and to study the variation in fluctuations between simple molecules like the DNA (which have only a secondary structure) to molecules with higher order structures and packaging. We have developed a new technique - Thermal Fluctuation Spectroscopy (TFS) to study these fluctuations. The technique of Thermal Fluctuation Spectroscopy (TFS) is a combination of microcalorimetry and noise measurement techniques. The combination of these two powerful techniques has never been exploited before. In this technique any energy exchange between sample and the substrate is reflected as a thermal fluctuation of the substrate. The system resolution is few parts per billion (ppb) and fluctuations in energy ~ 100nJ (which correspond to temperature fluctuations ~ K) can be measured. Chromatin is the basic building block of chromosome and this thesis focuses on the constituents this fundamental building block - DNA, histones and nucleosomes. Heteropolymeric dsDNA shows extremely large non-Gaussian fluctuation around its melting temperature. For homopolymeric DNA the fluctuations during denaturation are smaller. The thermal fluctuation during denaturation of a heteropolymer in buffer is several orders larger than when the DNA is on a substrate while that for a homopolymer is comparable in both cases. Our measurements established that heteropolymeric dsDNA denaturation occurs in two stages. Initially, at around 330 K, bubbles are formed in the AT rich regions. At higher temperatures, the GC rich regions binding them denature in a cooperative transition causing extremely large fluctuations. TFS on histone monomers showed that H1 monomer shows an increase in thermal fluctuation in the temperature range studied, while the core histones did not. We infer that this is due to the fact that the core histones may not be properly folded when they exist as monomers. It was seen that H1 crosses an energy barrier of 17 kcal/mol to go from its native to denatured state. The transition was kinetically driven with a fixed barrier till 352 K. At 352K, the barrier softened by ~ 1 kcal/mol leading to faster denaturation. The core histones when assembled as dimers/oligomers showed an increase in fluctuation at temperatures below 350 K. The assembling of these histones and DNA into a mononucleosome causes a very large increase in fluctuation over the entire temperature range studied. TFS showed that the fluctuation during mononucleosome denaturation was much larger than a simple sum of the fluctuations of its constituents. From the data we were able to identify that the denaturation starts with dissociation and unfolding of the core histones and the denaturation of AT rich regions of the DNA which leads to the breaking of some of the histone-DNA contacts. At higher temperatures the linker histone H1 and the GC rich regions of the DNA denature, leading to a collapse of the entire nucleosome structure. The broadness of the transition region (the fact that the fluctuation is large over the entire temperature range) was attributed to the presence of different types of contacts and interactions (with different energies) stabilizing the nucleosome structure. The nucleosome was found to favour large energy jumps over smaller ones indicating that the denaturation has an element of cooperativity involved. Using TFS we have been able to determine the fluctuations involved in the denaturation of biomolecules like DNA, histones and nucleosomes. The energy barriers to denaturation have been determined. We have also been able to give models for the denaturation of these biomolecules. We have also shown that it is possible to study enzymatic digestion using TFS. Thus, the technique of TFS is a viable tool for the study of fluctuations in reactions, in biomolecules, during transitions and in any process where there is an energy exchange involved.

Denaturation and renaturation is indispensable for the biological function of nucleic acids in many cellular processes, such as for example transcription for the synthesis of RNA and DNA replication during cell division. However, the reversible hybridisation of complementary nucleic acids is equally crucial in nearly all molecular biology technologies, ranging from nucleic acid amplification technologies, such as the polymerase chain reaction, and DNA biosensors to next generation sequencing. For nucleic acid amplification technologies, controlled DNA denaturation and renaturation is particularly essential and achieved by cycling elevated temperatures. Although this is by far the most commonly used method, the management of rapid temperature changes requires bulky instrumentation and intense power supply. These factors so far precluded the development of true point-of-care tests for molecular diagnostics. This Thesis explored the possibility of using electrochemical means to control reversible DNA hybridisation by using electroactive intercalators. First, fluorescence-based melting curve analysis was employed to gain an in depth understanding of the reversible process of DNA hybridisation. Fundamental properties, such as stability of the double helix, were investigated by studying the effect of common denaturing agents, such as formamide and urea, pH and monovalent salt concentration. Thereafter, four different electroactive intercalators and their effect on the thermodynamic stability of duplex DNA were screened. The intercalators investigated were methylene blue, thionine, daunomycin and adriamycin. Absorbance-based melting curve analysis revealed a significant increase of the melting temperature of duplex DNA in the presence of oxidised daunomycin. This was not observed in the presence of chemically reduced daunomycin, which confirmed the hypothesis that switching of the redox-state of daunomycin altered its properties from DNA binding to non-binding. Accordingly this altered the thermodynamic stability of duplex DNA. The difference in the stability of duplex DNA, as a direct result of the redox-state of daunomycin, was exploited to drive cyclic electrochemically controlled DNA denaturation and renaturation under isothermal conditions. This proof-of-principle was demonstrated using complementary synthetic 20mer and 40mer DNA oligonucleotides. Analysis with in situ UV–vis and circular dichroism spectroelectrochemistry, as two independent techniques, indicated that up to 80 % of the duplex DNA was reversibly hybridised. Five cycles of DNA denaturation and renaturation were achieved and gel electrophoresis as well as NMR showed no degradation of DNA or daunomycin. As no extreme conditions were implicated, no covalent modification of DNA was required and isothermal conditions were kept, this finding has great potential to simplify future developments of miniaturised and portable bioanalytical systems for nucleic acid-based molecular diagnostics.

The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as riboflavin derivatives, using a tightly-bound flavin mononucleotide (FMN) cofactor. It has important environmental and clinical implications. Previous studies have focused on elucidating NRs catalytic mechanism and solving its crystal structure. In this dissertation work, we first develop and implement new strategies for labeling NR with stable isotopes, and report a completely re-designed protocol for NRs purification. Then we report the successful assignment of over half of NRs backbone resonances using 3d-NMR methods. The most significant observation is that we find a well-resolved 2d 1H-15N HSQC NMR spectrum is obtained at 37°C for NR, while the HSQC at 4°C is poorly-dispersed and comprised of sharp overlapped peaks. Thus, it would appear that NR denatures at 4°C. However, as we demonstrate, the non-covalently-bound FMN cofactor is not released at the lower temperature, based on retention of the native flavin visible-CD spectrum. Similarly, far-UV CD spectroscopy shows that the protein retains full secondary structural content at 4C. In addition, near-UV CD and Fluorine-19 NMR experiments demonstrate that under completely native conditions (neutral pH, no additives) NR maintains a high degree of tertiary structure and well-defined hydrophobic side-chain packing, ruling out the possibility of a molten-globule state. Thus, our studies report strong evidence that the dramatic low-temperature (low-T) transition near 20°C observed by NMR is not the result of protein structural changes, but rather, we propose that NR exists as an ensemble of rapidly inter-converting structures, at lower temperature, only adopting a well-defined unique structure above 20°C. Both saturation-transfer from water and solvent proton-exchange measurements support our proposed model in which the unique high-T structure is favored entropically, by release of water molecules; on the other hand, the fluxional low-T state incorporates greater water solvation at 4°C. In the latter part of this study, we present preliminary data suggesting that the flexibility implied by easy water-access to the loosely-structured state plays a role in accommodating binding of diverse substrates. Therefore, the fluxional low-T state may be functionally important. A possible direct link between the enzyme dynamics and its catalytic activity will be an area of future investigation.

Spores of foodborne pathogens such as Clostridium botulinum, Clostridium perfringens and Bacillus cereus are widely distributed in nature. Presence of those spores in food products, particularly C. botulinum spores in vacuum packed, ready-to-eat low-acid products, is a great safety concern. The research here described is a first effort towards understanding the role of the spore coat proteins in the inactivation of bacterial spore using high pressure processing. This study proposes a coat protein solubilization methodology using non-ionic detergents minimizing protein damage and compatible with spectroscopy methods. The methodology developed here was compared with approaches proposed in the literature with respect to protein yield, protein fractions identified, amino acid composition and suitability with spectroscopy techniques for the further analysis of coat proteins. Bacillus subtilis ATCC 6633 spore coat proteins were solubilized (n=3) using octyl-β-D-glucopyranoside (OGP) at room temperature and urea/sodium dodecyl sulphate (UDS) at 37C and 70C. Analysis of variance (ANOVA) showed no significant (95% confidence) differences between the three repetitions of the three spore coat protein solubilization methods. Protein yield was significantly larger (95% confidence) when using UDS at 70C as compared to UDS at 37C. OGP gave the lowest protein yield but allowed circular dichroism (CD) analysis of the spore coat protein solution with minimum blank signal. SDS-PAGE revealed that the UDS-70C coat protein solutions consisted of five major and six minor proteins ranging 6 to 65 kD while the OGP solution appear to consist of four major and nine minor bands in the same mw range. Amino acid analysis of the protein extracted by the OGP method was conducted using reverse phase HPLC (RP-HPLC) and compared with published information. The OGP spore coat protein solution showed a higher proportion of aspartate, glutamate, alanine and tyrosine. Pressure, heat and time effects were studied on spore coat proteins obtained from untreated and pressure-treated B. subtilis ATCC 6633 spores. Pressure treatments of spores, and of extracted spore coat protein solutions, at 50 kpsi (345 mPa) and 85 kpsi (586 mPa) for 10 and 30 min at constant 85C along with appropriate heat- and pressure-only controls and untreated sample, were used to study the effect of pressure, heat and time on spore coat proteins. Both spore coat protein solubilization procedures showed a significant reduction in protein yield for pressure-only, heat-only and pressure/heat treated spores when compared with untreated spores. When OGP-solubilized proteins from untreated spores were pressure treated, SDS-PAGE profile showed an increasing overall band intensity with increasing pressure and time. In the case of protein solution obtained from pressure-treated spores the electrophoretic pattern showed the loss of higher molecular weight proteins. The significance of this study is that for the first time we have observed extensive changes on spore coat proteins caused by pressure, as well as heat treatments. Future studies will examine what is the probable physiological role of the proteins damaged by these physical treatments. An advantage of the protein solubilization here developed will allow the application of spectroscopy techniques to characterize changes in spore coat proteins. / Graduation date: 2003

SETD2/HYPB (SET domain-containing protein 2 / Huntingtin yeast partner B) is the predominant lysine methyltransferase in mammals that mediates histone H3 lysine-36 (H3K36) trimethylation, which is associated with transcription elongation and RNA splicing. SETD2 is further implicated in p53 function, vascular development, cancer progression and, through Huntingtin-interaction, Huntington's disease. Although different transcripts and putative protein isoforms have been detected previously, their identity, function and significance have not been rigorously investigated. This thesis aims to identify and characterise endogenous transcripts and protein isoforms of SETD2 in mouse fibroblasts. Affnity-purified N- and C-terminal antibodies specifically detected the &TildeTilde; 290 kDa methyltransferase (p290^SETD2), verified by RNAi, in addition to N terminal-specific &TildeTilde; 120 kDa protein, and C terminal-specific forms at &TildeTilde; 140 and &TildeTilde; 66 kDa (p66), which all appeared too stable to deplete by transient siRNA transfection. Conserved in human and mouse cells, immunodetection of p66 exhibited unusual requirement for denaturation with urea at 95°C. Subcellular fractionation revealed distinct extraction properties of putative isoforms and facilitated partial purification of p66 for proteomic analysis. Co-fractionation and co migration by two-dimensional gel electrophoresis of p66 detected by two independent C terminal antibodies suggested it represents a novel C terminal-specific isoform. Reverse transcription−PCR and DNA-sequencing demonstrated the existence of multiple, alternatively-spliced Setd2 transcripts that plausibly generate truncated proteins. A transcript variant containing a novel complete open-reading-frame, consistent for p66 generation, was identified. Its ectopic expression in mouse fibroblasts produced a distinct SETD2 isoform, whose physical and extraction characteristics were studied in comparison with endogenous immunoforms. In summary, this thesis demonstrates that multiple alternatively-spliced transcripts arise from the Setd2 gene, consistent with immunodetection of several C- and N-terminal-specific putative SETD2 isoforms, additional to the H3K36 methyltransferase. Verification of these isoforms by independent methods would have implications for proposed interactions and function of SETD2 in transcription, epigenetics, cancer development and Huntington’s disease.

Diploma thesis is focused on creation of educational materials supporting the education of biochemistry, specifically amino acids and proteins, at secondary level. At first the analysis of Czech chemistry textbooks concerning the two topics - amino acids and proteins was performed. The major problems found were related to the insufficient graphical representation of biomolecules, unsatisfactory motivational components and insufficient integration of the topic with biology or everyday life experience. The supporting educational materials, presented in this work, supplement the widely used chemistry textbooks. The materials includes a graphic oriented presentation, interactive animations demonstrating the process of denaturation and precipitating of proteins at macroscopic and molecular level, poster presenting the structural formulas of standard amino acids, 3D models of selected proteins, additional texts supporting the current topics concerning amino acids and proteins and the laboratory protocols for students. The presented support materials were evaluated at the secondary school conditions. They were tested in one class and the improvement of student's understanding of the topic was compared to the second class employing only the classical educational methods. The comparison of the results of...

Este estudo tem o objetivo de analisar a relação entre educação e o processo de desnaturação do homem na obra Emílio ou Da Educação de Jean-Jacques Rousseau. Pretende-se, com isso, examinar as distintas formas de desnaturação concebidas por Rousseau em suas obras. Para tanto, investigar-se-á primeiramente as ideias de educação e de natureza no século XVIII, a fim de se esclarecer as relações que se estabeleciam entre ambas as ideias. Em seguida, buscar-se-á compreender a concepção de Rousseau sobre a educação pública. Segundo o autor, as boas instituições sociais são as que melhor sabem desnaturar o indivíduo, fazendo do homem um cidadão. Neste caso, o papel da educação pública consiste em promover uma boa desnaturação do homem, ou uma desnaturação num sentido positivo. Entretanto, o que se verifica na obra de Rousseau Discurso sobre a desigualdade entre os homens é um processo de desnaturação de toda espécie humana, mas no sentido negativo do termo. O que ocorreu na história da espécie foi o que se pode chamar de uma má desnaturação do homem, pela qual foram anulados seus atributos naturais, como a liberdade, o amor de si e a consciência, associados, por sua vez, aos progressos da sociedade. Dessa forma, o homem, ao tornar-se sociável, passa a sofrer uma contradição interna entre suas inclinações naturais, que subsistem, e seus deveres de homem sociável. A má desnaturação se caracteriza então por ser imperfeita, ou incompleta, visto que engendra esta contradição no homem entre natureza e sociedade. Impõe-se assim, como questão da educação no Emílio, resolver essa contradição no homem, e formá-lo de modo que ele seja para si e para os outros. Procurar-se-á assim examinar a formação do personagem Emílio, a fim de elucidar como se estabelece, neste caso, o processo de desnaturação, visto que este processo significa alguma forma de socialização, seja a da má ou a da boa desnaturação. Emílio deverá ser formado primeiramente para si, portanto, de acordo com a natureza, mas a partir de uma determinada idade ele será formado para os outros, ou seja, para se tornar homem sociável, e de certo modo um cidadão. Sua educação será composta então pelos elementos que constituem as diferentes formas de desnaturação, tal como se apresentam no pensamento de Rousseau. Pretende-se assim analisar o processo de desnaturação efetuado pela educação de Emílio, que o mantém contudo de acordo com a natureza, tornando-o homem, mas também cidadão. / This study aims to analyze the bonds between education and denaturation process of man in Jean-Jacques Rousseaus Emile, or on Education. By doing so, this research intends to assay the different forms of denaturation conceived by Rousseau in his writings. Thereby, we will begin by investigating the ideas of education and nature in the eighteenth century, in order to clarify the links established between these two ideas. Thereafter, we will seek to understand Rousseaus conception of public education. According to Rousseaus thought, good social institutions are the ones that can best denaturalize the individuals, by making them citizens. In this manner, the role of public education is to promote a good denaturation of man, or a denaturation in a positive sense. However, Rousseaus Discourse on Inequality shows a process of denaturation of the whole human race, but in the negative sense of the term. Throughout the history of the species occurred what we may call a bad denaturation of man, by which his natural characteristics such as freedom, the love of self and consciousness, were nullified, associated, in their turn, with the progress of society. Thus, man, by becoming sociable, starts suffering an inner contradiction between his natural inclinations, which still endure, and his duties as a socialized man. The bad denaturation is characterized by being imperfect, or incomplete, for it engenders this contradiction in man between nature and society. The issue of education in Emile is, then, to deal with this contradiction in man, and to educate him so that he becomes a man for himself and for the others. We will thus consider the education of the character named Emile, in order to elucidate how the denaturing process is established in this case, since this process means some form of socialization, be it the bad or good denaturing. Firstly, Emilio has to be educated for himself, therefore, in accordance with nature; meanwhile, from a given age on he will be formed for the others, that is, to become a sociable man and in a certain way a citizen. His education will then be composed by the elements that constitute different forms of denaturation, as they are presented in Rousseau\'s thought. Therefore, we intend to research the denaturing process carried out in Emilios education, a fulfilment that keeps him, however, in agreement with nature, making him a man, but also citizen.