L'adaptation à l'environnement est essentielle pour la survie de tout organisme, des bactéries à l'Homme. Pour s'adapter rapidement aux milieux extrêmement variés et aux fluctuations environnementales, les procaryotes ont adopté des systèmes élaborés, capables de détecter et de réagir aux molécules vitales ou toxiques à leur développement. Outre les facteurs de stress tels que température, pH, la cellule doit s'adapter aux variations de concentration de nutriments et de gaz diatomiques. En effet, la modification d'un ou plusieurs paramètres de l'environnement déclenche une altération de l'expression des gènes, permettant des changements dans la composition protéique et ainsi une adaptation du métabolisme aux nouvelles conditions. Un paramètre environnemental particulièrement essentiel est le taux d'oxygène. Le passage à la vie limitée en oxygène, ou inversement, a été particulièrement étudié chez les bactéries. Celles-ci disposent de plusieurs chaînes de transporteurs d'électrons et utilisent à tout moment celle qui leur est la plus favorable sur le plan énergétique. En présence d'oxygène, accepteur d'électrons le plus favorable, les bactéries utilisent la respiration oxygènée, qui se caractérise par la synthèse d'enzymes du type cytochrome oxydase, alors qu'en absence d'oxygène ou en condition de basse pression d'oxygène (ou hypoxie), elles mettent en place la respiration par le nitrate, caractérisée par la synthèse de quinones respiratoires comme intermédiaires (Pelmont J., 1993). Le transporteur final de la voie " nitrate " est la nitrate réductase, enzyme clé inhibée par l'oxygène. La présence d'oxygène diatomique impose donc aux bactéries de profonds changements de leur métabolisme, soit parce qu'elles doivent réajuster leur système de production d'énergie, soit parce qu'elles doivent lutter contre la toxicité de l'oxygène ; pour cela elles disposent de tout un arsenal enzymatique. La connaissance des processus biochimiques par l'intermédiaire desquels ces adaptations sont réalisées est essentielle à la compréhension du fonctionnement cellulaire.

GUIMARÃES, Wellinson Gadêlha. FixL Híbrida da Bactéria Rhizobium etli: Estudos Conformacionais e de Estabilidade. 2016. 74 f. Dissertação (Mestrado em Química)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-03-27T11:47:49Z No. of bitstreams: 1 2016_wgguimaraes.pdf: 2399264 bytes, checksum: 6b01ad2806daa748fbbbddeee1ed0c0d (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-03-27T22:56:19Z (GMT) No. of bitstreams: 1 2016_wgguimaraes.pdf: 2399264 bytes, checksum: 6b01ad2806daa748fbbbddeee1ed0c0d (MD5) / Made available in DSpace on 2017-03-27T22:56:19Z (GMT). No. of bitstreams: 1 2016_wgguimaraes.pdf: 2399264 bytes, checksum: 6b01ad2806daa748fbbbddeee1ed0c0d (MD5) Previous issue date: 2016 / Heme-based sensors are a class of hemeproteins that studies are relatively recent. Heme-based sensor are capable to reversibly bind to molecules such as O2, CO and NO ligands through iron atom, leading to conformational changes that govern adptative responses, make them actives or inactives to a response. FixL is a histidine kinase found in several bacteria Rhyzobium, being involved in microaerobic respiration and nitrogen metabolism processes. This protein has been extensively studied and is considered a model system to heme-based sensors that are histidine kinases. Its mechanisthic elucidation may help to understand many similar systems. In this work protein FixL from Rhyzobium etli, until now one only of genre that was studied, was produced and purified aiming to investigate conformational and stability aspects. Therefore, it was used Circular Dichroism (CD) to estimate the kind of secondary structure in FixL in a active state. It was revealed that it is majority organized on α-helices. After, it was investigated the possible conformational changes that FixL protein may have when its activity is changed due to be bounded to a signalizing ligand. Interesting results obtained suggest the potential use of CD technique and electronic spectroscopy to assess structural changes from an active state to inactive one that apparently occur by changing the tertiary structures. The results suggest that the heme interactions with nearby protein side chains are involved in loss of activity from FixL to bind O2 or CN-. Finally, stability studies by thermal and chemical denaturation showed that FixL is less stable than many other heme proteins, and that there are significant differences in stability between the active state and the inactive state, particularly with regard to chemical denaturation, although not had significant differences in the thermal stability. These differences in stability were explained in the light of a model in which the protein becomes more compact when it is enzymatically active by interaction inter / intra domains. / Heme proteínas sensoras (HPS) são uma classe de heme proteínas cujos estudos são relativamente recentes. As HPS são capazes de se ligar reversivelmente a moléculas como O2, CO e NO por meio do ferro do seu grupo heme, o que leva a alterações estruturais que governam as respostas adaptativas, tornando-as ativas ou inativas para uma resposta. A FixL é uma histidina quinase encontrada em várias bactérias do gênero Rhizobium, estando envolvida nos processos de respiração microaeróbica e no metabolismo do nitrogênio. Esta proteína tem sido bastante estudada, sendo considerada um sistema modelo para hemeproteínas sensoras que desempenham função histidina quinase, e cuja elucidação mecanística pode ajudar a entender diversos sistemas semelhantes. Neste trabalho foi produzida e purificada a proteína FixL da bactéria Rhyzobium etli, única do gênero estudada até agora, tendo como finalidade investigar aspectos conformacionais e de estabilidade. Desta forma, empregou-se espectroscopia de dicroísmo circular (CD) para estimar os tipos de estrutura secundária desta proteína em seu estado nativo, sendo revelado que sua estrutura se encontra majoritariamente na forma de α-hélices. Posteriormente, foram investigadas as possíveis alterações conformacionais que a proteína sofre por ocasião da mudança na sua atividade enzimática provocada por ligantes sinalizadores. Os interessantes resultados obtidos ilustram a potencialidade da técnica de CD e espectroscopia eletrônica em avaliar alterações estruturais de um estado ativo para inativo que ocorrem aparentemente através da mudança das estruturas terciárias. Os resultados sugerem que as interações do grupo heme com as cadeias protéicas laterais próximas estão envolvidas na perda de atividade da FixL ao se ligar ao O2 ou ao CN-. Por fim, os estudos de estabilidade por desnaturação térmica e química mostraram que a FixL é menos estável que diversas outras heme proteínas, e que existem diferenças significativas de estabilidade entre o estado ativo e o estado inativo, particularmente quanto à desnaturação química, apesar de não haverem diferenças significativas em relação à estabilidade térmica. Essas diferenças na estabilidade foram explicadas à luz de um modelo em que a proteína se torna mais compacta quanto está enzimaticamente ativa através de interações inter/intra domínios.

In this thesis, molecular dynamics simulations, molecular docking, and homology modeling methods have been used in combination to design possible inhibitors as well as to study the structural changes and function of target proteins related to diseases that today are in the spotlight of drug discovery. The inwardly rectifying potassium (Kir) channels constitute the first target in this study; they are involved in cardiac problems. On the other hand, tensin, a promising target in cancer research, is the second target studied here. The first chapter includes a brief update on computational methods and the current proposal of the combination of MD simulations and docking techniques, a procedure that is applied for the engineering of a new blocker for Kir2.1 ion channels and for the design of possible inhibitors for Tensin. Chapter two focuses in Kir ion channels that belong to the family of potassium-selective ion channels which have a wide range of physiological activity. The resolved crystal structure of a eukaryotic Kir channel was used as a secondary structure template to build the Kir-channels whose crystallographic structures are unavailable. Tertiapin (TPN), a 21 a.a. peptide toxin found in honey bee venom that blocks a type of Kir channels with high affinity was also used to design new Kir channel blockers. The computational methods homology modeling and protein-protein docking were employed to yield Kir channel-TPN complexes that showed good binding affinity scores for TPN-sensitive Kir channels, and less favorable for Kir channels insensitive to TPN block. The binding pocket of the insensitive Kir-channels was studied to engineer novel TPN-based peptides that show favorable binding scores via thermodynamic mutant-cycle analysis. Chapter three is focused on the building of homology models for Tensin 1, 2 and 3 domains C2 and PTP using the PTEN X-ray crystallographic structure as a secondary structure template. Molecular docking was employed for the screening of druggable small molecules and molecular dynamics simulations were also used to study the tensin structure and function in order to give some new insights of structural data for experimental binding and enzymatic assays. Chapter four describes the conformational changes of FixL, a protein of bradyrhizobia japonicum. FixL is a dimer known as oxygen sensor that is involved in the nitrogen fixation process of root plants regulating the expression of genes. Ligand behavior has been investigated after the dissociation event, also the structural changes that are involved in the relaxation to the deoxy state. Molecular dynamics simulations of the CO-bound and CO-unbound bjFixL heme domain were performed during 10 ns in crystal and solution environments then analyzed using Principal Component Analysis (PCA). Our results show that the diffusion of the ligand is influenced by internal motions of the bound structure of the protein before CO dissociation, implying an important role for Arg220. In turn, the location of the ligand after dissociation affects the conformational changes within the protein. The study suggests the presence of a cavity close to the methine bridge C of the heme group in agreement with spectroscopic probes and that Arg220 acts as a gate of the heme cavity.