A estabilidade térmica em função do pH para três formas da hemoglobina extracelular gigante do anelídeo Glossoscolex paulistus (HbGp), monitorada atraves de DSC, CD e absorção óptica, e estudada no presente trabalho. Estes estudos possibilitaram a determinação de parâmetros importantes do processo de desnaturação e dissociação da proteína oligomerica em pH ácido, neutro e alcalino. A HbGp se mostrou mais estável no pH ácido do que em pH neutro e alcalino. No meio alcalino a HbGp sofre dissociação oligomérica gerando subunidades tais como o dodecâmero, o trímero e o monômero. Além disso, as técnicas de DSC, dicroísmo circular (CD) e absorção óptica permitiram o monitoramento da desnaturação da estrutura protéica global, da estrutura secundária e do centro ativo da HbGp, em função da temperatura. Por DSC foi determinado que o mecanismo do processo de desnaturação térmica da HbGp é irreversível. As variações de entalpia calorimétrica, ΔHcal, e de van Hoff, ΔHvH, nas formas oxi-, meta- e cianometa-HbGp são bem distintas, em todos os pHs estudados, indicando que o processo de desnaturação é bastante complexo, sugerindo que o pico de transição deve ser composto por varias transições. A ordem de estabilidade apresentada pela HbGp em termos dos valores de temperatura de transição (Tm) foi a seguinte: cianometa- > oxi- > meta- no intervalo de pH 5,0 a 8,0. Os valores de ΔHcal no pH 7,0 para a oxi-HbGp, meta-HbGp e cianometa-HbGp foram de 25 ± 4, 20 ± 2 e 56 ± 4 MJ/mol, respectivamente. Os valores de energia de ativação (Ea) obtidos no pH 7,0 para a oxi- e cianometa-HbGp foram de 673 ± 99 e 780 ± 105 KJ/mol, e no pH 8,0 de 897 ± 106 e 850 ± 201 KJ/mol, respectivamente. Esses valores de energia de ativação são condizentes com os reportados na literatura para outras hemoglobinas. Nos estudos realizados por CD a oxi-HbGp forma hemicromo no pH 6,0 e 7,0, em temperaturas superiores a 40 °C, e se dissocia em meio alcalino. A oxi-HbGp apresenta temperatura crítica (Tc) nas regiões das hélices-α e do grupo heme praticamente idêntica nos vários pHs estudados. A cianometa-HbGp possui maior quantidade de estrutura secundária do que a oxi-HbGp, e maiores valores de temperatura crítica (Tc), sendo bem mais estável que a oxi-HbGp, assim como o observado por DSC. Por absorção óptica o comportamento térmico da HbGp é similar ao do CD, sendo observado ainda, além da formação de hemicromo, a presença de espécies pentacoordenadas no pH neutro e alcalino. / The thermal stability as a function of the pH, for three forms of the extracellular giant hemoglobin of the annelid Glossoscolex paulistus (HbGp) was monitored by DSC, CD and optical absorption in the present work. These studies allowed the determination of important parameters characterizing the denaturation and dissociation at acid, neutral and alkaline pH values. HbGp was shown to be more stable in acid pH as compared to neutral and alkaline pH values. In alkaline medium, HbGp presents oligomeric dissociation generating smaller subunits such as the dodecamer, the trimer and the monomer. Besides that, the techniques of the DSC, circular dichroism (CD) and optical absorption spectroscopy allowed to monitor, respectively, the denaturation of the global protein structure, of the secondary structure and of the active center of the hemoglobin, as a function of the temperature. By DSC it was determined that the mechanism of the thermal denaturation of the HbGp is irreversible. The variations of calorimetric and van Hoff enthalpies, in the oxy- and cyanomet-HbGp forms, are quite different, for all studied pH values, indicating that the process of denaturation is complex, characterized by a transition peak composed by several contributions. The order of stability presented by the HbGp in terms of the transition temperature values (Tm) was the following: cyanomet-> oxy- for pH from 5.0 to 8.0. The values of ΔHcal at pH 7.0 for the oxy-HbGp, met-HbGp and cianomet-HbGp were 25 ± 4, 20 ± 2 and 56 ± 4 MJ/mol, respectively. The activation energy values (Ea) obtained at pH 7.0 for the oxy- and cyanomet-HbGp were 673 ± 99 and 780 ± 105 KJ/mol, and at pH 8.0 they were 897 ± 106 and 850 ± 201 KJ/mol, respectively. Those energy values are consistent with data reported in the literature for other hemoglobins. In the studies carried out by CD for oxy-HbGp formation of hemichrome was observed at pH 6.0 and 7.0, at temperatures above 40 °C. In alkaline medium the oligomeric dissociation is observed. Oxy-HbGp presents critical temperatures (Tc), which are practically identical in the spectral regions of the polypeptide and of the heme groups for all studied pH values. The cyanomet-HbGp own larger quantity of secondary structure than oxy-HbGp, and higher values of critical temperatures (Tc), being more stable than oxy-HbGp, in agreemente with DSC data. Optical absorption spectroscopy shows thermal behavior of HbGp similar to that observed by CD. Besides the formation of the hemichrome species upon heating, the presence of penta-coordinate species at neutral and alkaline pH values was observed.

The diploma thesis deals with the preparation, crosslinking and characterization of collagen films from various animal sources. Collagen from pig, Tilapia, horse, cow and crocodile was used to prepare collagen films. Chemical crosslinking agents EDC/NHS and Lyofix were used to crosslink the prepared films. In the experimental part, differential scanning calorimetry (DSC) method was optimized to determine the denaturation temperature of individual collagen films before and after crosslinking. In addition, the ability of films to swell and degrade has been analyzed. The presence of characteristic groups present in the collagen structure was verified using infrared spectroscopy. The sample morphology was analyzed using Scanning Electron Cryomicroscopy (Cryo-SEM). The results show that EDC/NHS is a better collagen crosslinking agent compared to Lyofix. The best source for the preparation of thermally stable films is piggy collagen, whose denaturation temperature after crosslinking with EDC/NHS was about 69 °C and could represent more than adequate substitution for cow collagen, which is currently most used in the field of tissue engineering and food industry.

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.

L'amélioration de la qualité nutritionnelle des produits carnés cuits nécessite une meilleure compréhension des changements physicochimiques des protéines induits au chauffage. Ce travail porte sur l'analyse des mécanismes à l'origine des changements d'état des protéines afin de développer un modèle stoechio-cinétique de prédiction de l'effet de la composition et de la température sur ces changements. Un modèle expérimental, représentant l'environnement physicochimique du tissu musculaire (pH et force ionique), a permis de quantifier l'incidence spécifique de la chaleur, de la composition en fibres, en oxydants (fer, peroxyde d'hydrogène et vitamine C) et en antioxydants (enzymatiques, vitaminique et peptidique) sur l'oxydation, la dénaturation thermique et l'agrégation des protéines. Le modèle stoechio-cinétique est constitué de 43 réactions, représentant l'ensemble des phénomènes mis en jeu dans le modèle expérimental : chimie de Fenton, attaques radicalaires des acides aminés et dénaturation thermique. La résolution du système d'équations différentielles permet de calculer les concentrations des composés au cours du chauffage ; 3 constantes de vitesse inconnues ont été ajustées à partir des cinétiques expérimentales. Les résultats expérimentaux montrent : (1) un effet synergique des oxydants et du chauffage sur les oxydations, (2) une incidence négligeable des oxydants sur la dénaturation thermique et l'agrégation, (3) une sensibilité accrue des protéines de fibres α-white aux oxydations et à la dénaturation thermique par rapport à celles de fibres β-red et (4) un important effet de la nature des oxydants et des antioxydants sur les taux d'oxydation. Les prédictions du modèle stoechio-cinétique permettent de reproduire les tendances expérimentales. En partant de cette base, les modèles expérimentaux et mathématiques pourront être complexifiés progressivement pour avoir un outil prédictif de la qualité nutritionnelle des viandes cuites. / Improving the nutritional quality of cooked meat products needs a better understanding of protein physicochemical changes induced by heating. This study aims to analyse the mechanisms responsible to protein state changes, in the goal to develop a predictive stoichio-kinetic model of effect of composition and temperature on these changes. An experimental model which represent the physicochemical environment of meat tissue (pH and ionic strength) allowed to quantify the specific effect of heating, composition in fibres, in oxidants (iron, hydrogen peroxide and vitamin C) and in antioxidants (enzymes, vitamins and peptides) on oxidations, thermal denaturation (hydrophobicity) and aggregation of proteins. The stoichio-kinetic model is composed of 43 reactions which represent all phenomenon involved in the experimental model : Fenton chemistry, radical attack on amino acids and thermal denaturation. A system of differential equation solver allows to determine the concentration of compounds during heating ; 3 unknown rate constants were adjusted with experimental kinetics. Experimental results show : (1) a synergistic effect of oxidants and heating on protein oxidation, (2) a negligible impact of oxidants on thermal denaturation and aggregation (3) a significant higher sensitivity to oxidation and thermal denaturation of protein from α-white than those from β-red, (4) an important effect of the composition in oxidants and antioxidants on the protein oxidation levels. Stoichio-kinetic model predictions reproduce experimental tendencies. From this base, experimental and stoichio-kinetic models could be progressively complexified to obtain a predictive tool of nutritional quality of meat.

SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.

SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.

The majority of structural studies on DNA have been carried out using fibre diffraction, while studies of its dynamics and thermal behaviour have been mainly performed in solution. When the DNA double helix is heated, it exhibits local separation of the two strands that grow in size with temperature and lead to their complete separation. This work has investigated various aspects of this phenomenon. The experiments reported in this thesis were carried out on films of oriented fibres of DNA prepared with the Wet Spinning Apparatus. Thus, sample preparation and characterisation are essential parts of the research. The structures of two forms of DNA, A and B, have been explored as a function of relative humidity at fixed ionic conditions. A method to eliminate traces of ever-present B-form contamination in A-form samples was established. The high orientation of the DNA molecules within the samples allowed us to investigate dynamical fluctuations and the melting transition of DNA using neutron scattering, which can provide the spatial information crucial to understand a phase transition, probing the static correlation length along the molecule as a function of temperature. The transition has been investigated for A and B-forms in order to understand its dependence on molecular configuration.Furthermore, after the first melting, denatured DNA films show typical glass behaviour. Their thermal relaxation has been explored using calorimetry.Neutron and X-ray inelastic scattering (INS and IXS) were used in the past to measure longitudinal phonons in fibre DNA, and the results shown disagreement. Recent INS measurements supported with phonon simulations have been crucial to understand the different dispersion curves reported to date. Experiments using INS and IXS have been carried out to continue with this investigation. Attempts to observe the transverse fluctuations associated to the thermal denaturing of DNA, never experimentally investigated before, have been made.

No description available.

La majorité des études structurales sur l’ADN avaient été réalisées par diffraction sur des fibres tandis que ses propriétés dynamiques thermiques avaient été étudiées en solution. Lorsque la double hélice d’ADN est chauffée elle présente des séparations locales des deux brins, dont la taille augmente avec la température jusqu’à la séparation complète des brins. Ce travail étudie différents aspects de ce phénomène. Les expériences présentées dans cette thèse ont été réalisées sur des films formés de fibres orientées d’ADN préparés par la méthode du ”filage humide“. La préparation et la caractérisation des échantillons en deux formes A et B de l’ADN ont constitué une partie importante de la recherche. Une méthode pour éliminer la contamination résiduelle de la forme B dans les échantillons de forme A a été mise au point. La bonne orientation des molécules d’ADN dans les échantillons nous a permis d’étudier les fluctuations dynamiques et la transition de dénaturation thermique de l’ADN par diffraction de neutrons, sensibles à la longueur de corrélation statique le long de la molécule en fonction de la température. La transition a été étudiée pour les formes A et B pour déterminer comment elle dépend de la conformation. De plus, après la première dénaturation thermique, les films d’ADN présentent un comportement typique d’un verre. Leur relaxation thermique a été étudiée par calorimétrie. La diffusion inélastique de neutrons et de rayons X (INS et IXS) avaient été utilisées antérieurement pour mesurer les phonons longitudinaux dans des fibres d’ADN, avec des désaccords entre les résultats. Des mesures INS récentes, complétées par des simulations, avaient été cruciales pour comprendre les différentes courbes de dispersion observées. Nous avons mené des expériences INS et IXS pour poursuivre cette analyse. Des tentatives pour observer les mouvements transversaux associés à la dénaturation thermique de l’ADN, jamais observés expérimentalement, ont également été faites. / The majority of structural studies on DNA have been carried out using fibre diffraction, while studies of its dynamics and thermal behaviour have been mainly performed in solution. When the DNA double helix is heated, it exhibits local separation of the two strands that grow in size with temperature and lead to their complete separation. This work has investigated various aspects of this phenomenon. The experiments reported in this thesis were carried out on films of oriented fibres of DNA prepared with the Wet Spinning Apparatus. Thus, sample preparation and characterisation are essential parts of the research. The structures of two forms of DNA, A and B, have been explored as a function of relative humidity at fixed ionic conditions. A method to eliminate traces of ever-present B-form contamination in A-form samples was established. The high orientation of the DNA molecules within the samples allowed us to investigate dynamical fluctuations and the melting transition of DNA using neutron scattering, which can provide the spatial information crucial to understand a phase transition, probing the static correlation length along the molecule as a function of temperature. The transition has been investigated for A and B-forms in order to understand its dependence on molecular configuration.Furthermore, after the first melting, denatured DNA films show typical glass behaviour. Their thermal relaxation has been explored using calorimetry.Neutron and X-ray inelastic scattering (INS and IXS) were used in the past to measure longitudinal phonons in fibre DNA, and the results shown disagreement. Recent INS measurements supported with phonon simulations have been crucial to understand the different dispersion curves reported to date. Experiments using INS and IXS have been carried out to continue with this investigation. Attempts to observe the transverse fluctuations associated to the thermal denaturing of DNA, never experimentally investigated before, have been made.