RHYTHM—a server to predict the orientation of transmembrane helices in channels and membrane-coils

RHYTHM is a web server that predicts buried versus exposed residues of helical membrane proteins. Starting from a given protein sequence, secondary and tertiary structure information is calculated by RHYTHM within only a few seconds. The prediction applies structural information from a growing data base of precalculated packing files and evolutionary information from sequence patterns conserved in a representative dataset of membrane proteins (‘Pfam-domains’). The program uses two types of position specific matrices to account for the different geometries of packing in channels and transporters (‘channels’) or other membrane proteins (‘membrane-coils’). The output provides information on the secondary structure and topology of the protein and specifically on the contact type of each residue and its conservation. This information can be downloaded as a graphical file for illustration, a text file for analysis and statistics and a PyMOL file for modeling purposes. The server can be freely accessed at: URL: http://proteinformatics.de/rhyth

MPlot—a server to analyze and visualize tertiary structure contacts and geometrical features of helical membrane proteins

MPlot is a webserver that provides a quick and easy way for structural biologists to analyze, visualize and plot tertiary structure contacts of helical membrane proteins. As input, experimentally determined or computationally modeled protein structures in PDB format are required. The automatic analysis concatenates in house tools to calculate cut-off dependent van der Waals contacts or crossing angles of transmembrane helices with third party tools to compute main chain or side chain hydrogen bonds or membrane planes. Moreover, MPlot allows new features and tools to be added on a regular basis. For that purpose, MPlot was embedded in a framework that facilitates advanced users to compose new workflows from existing tools, or to substitute intermediate results with results from their (own) tools. The outputs can be viewed online in a Jmol based protein viewer, or via automatically generated scripts in PyMOL. For further illustration, the results can be downloaded as a 2D graph, representing the spatial arrangement of transmembrane helices true to scale. For analysis and statistics, all results can be downloaded as text files that may serve as inputs for or as standard data to validate the output of knowledge based tertiary structure prediction tools

Modelação molecular/bioinformática estrutural da hemaglutinina do vírus influenza

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016O vírus influenza é responsável por vários surtos de gripe a nível mundial, como o recente surto causado pelo H1N1. A infeção começa com a ligação da proteína Hemaglutinina (HA) do envelope viral ao ácido siálico presente na membrana das células epiteliais do trato respiratório. O vírus é então internalizado por endocitose. No endossoma a diminuição de pH provoca alterações conformacionais da HA, levando por último à fusão das membranas. Neste trabalho reportamos um estudo de simulação molecular tanto da proteína HA, como também de dois péptidos específicos. O péptido de fusão (PF), que apresenta propriedades fusógenicas in vitro, sendo considerado o principal responsável pela fusão das membranas. E o péptido transmembranar (PT), responsável pela ligação da hemaglutinina ao envelope viral, que não possui estrutura conhecida, tanto a pH neutro como a pH de fusão. Nos estudos dos péptidos foram utilizados dois campos de força e níveis de detalhe distintos. Um campo de forças coarse-grain (MARTINI), mais rápido mas com menos detalhe, foi utilizado para simular a assemblagem do sistema e um campo de forças united-atom (GROMOS 54A7) para simular os resultados obtidos com um detalhe superior. Nos estudos da HA foi utilizado apenas o campo de forças coarse-grain, com a modificação para incluir redes elásticas (ELNEDIN). Com este estudo apresentamos estruturas, e algumas variações, para ambos os péptidos numa bicamada composta por 128 moléculas de dimiristoilfosfatidilcolina (DMPC). No caso do PT foi a primeira estrutura obtida com o C-terminal completo (46 resíduos) e também do subtipo H3. Foram ainda calculados os ângulos de inclinação e a posição, de cada resíduo, na membrana para ambos os péptidos estudados. No caso da HA o resultado principal foi o esclarecimento dos locais de contacto preferenciais entre esta e os lípidos (DMPC). Observou-se que, para além do domínio transmembranar, também existem resíduos à volta do local de ligação ao ácido siálico com afinidade para o DMPC.The influenza virus (IV) is responsible for worldwide flu outbreaks like the recent one due to H1N1. Infection starts with the binding of the membrane-bound virus Hemagglutinin (HA) to sialic acid at the surface of membranes from epithelial cells of the respiratory tract. The virus is then internalized into an endosome, where the low pH triggers conformational changes in HA, ultimately leading to membrane fusion. Here we report a molecular simulation study, not only of the HA, but also of two specific peptides. The fusion peptide (FP), which has fusogenic properties in vitro, and is considered to have a main role in the membrane fusion process. And the transmembrane peptide (TP), responsible for the connection between HA and the viral envelope, which has no known structure. We used a multi-scale approach in the studies with the peptides, by using two different force-fields with different levels of detail; a coarse-grain force field (MARTINI), which is faster but with less detail, to study the self assembling of the system, and an united-atom force-field (GROMOS 54A7), to study with more detail the results obtained using the MARTINI force-field. In the whole HA studies, we used the coarse-grain force-field only, but with the modification to include elastic networks (ELNEDIN). With this work we found some preferential structures and some variations, for both fusion and transmembrane peptides, in a bilayer composed by 128 molecules of dimyristoylphosphatidylcholine (DMPC). It is the first structure of the TP obtained with the complete C-terminus (46 residues) and also for the subtype H3. The tilt angles and the membrane position, of each residue, of both peptides studied were also calculated. In the case of the HA, the main result was to find that main sites of contact between this protein with lipids (DMPC). We found that apart from the transmembrane domain, the residues around the sialic acid binding site have also affinity to the DMPC lipids

GEM, una proteína con dominio GRAM, es un regulador negativo de la señalización por ABA durante la germinación en Arabidopsis thaliana

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-03-201

Etude de l'utilisation des acides aminés aspartate et asparagine dans la virulence de mycobacterium tuberculosis

La tuberculose tue plus d'un million de personnes par an à travers le monde et constitue la première cause de mortalité humaine due à un agent bactérien unique. L'agent étiologique de la tuberculose est Mycobacterium tuberculosis, un pathogène intracellulaire se répliquant à l'intérieur des macrophages dans une vacuole appelée le phagosome où l'accessibilité en nutriments est restreinte. Une meilleure compréhension des mécanismes par lesquels le bacille acquiert et assimile les nutriments de son hôte pourrait aider à développer de nouvelles stratégies pour combattre la tuberculose. Nous avons pu mettre en évidence que M. tuberulosis exploite les acides aminés aspartate et asparagine pour assurer sa croissance au cours de l'infection par l'utilisation des transporteurs AnsP1 et AnsP2 et de l'asparginase AnsA. Ce travail révèle, pour la première fois, que les acides aminés sont une source d'azote majeure utilisée par le bacille tuberculeux au cours de l'infection et ouvre la voie pour l'identification de nouveaux antituberculeux ciblant l'assimilation des acides aminés par ce pathogène au cours du cycle infectieux.Tuberculosis causes about 2 million dead per year in the worldwide and constitutes the first cause of human death due to a single bacterial pathogen. The etiologic agent of tuberculosis is Mycobacterium tuberculosis, a facultative intracellular pathogen who replicates inside macrophages in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here, we could demonstrate that M. tuberculosis exploits the amino acids aspartate and asparagine as a major nitrogen source to support growth during infection through the transporters AnsP1 and AnsP2 and the asparaginase AnsA. This work provide the evidence, for the first time, that amino acids are major nitrogen providers during host colonization and thus pave the way to identify new antituberculous compounds targeting assimilation of amino acids by the bacterium upon infection