RESUMO
SUMMARY: The Helmholtz Network for Bioinformatics (HNB) is a joint venture of eleven German bioinformatics research groups that offers convenient access to numerous bioinformatics resources through a single web portal. The 'Guided Solution Finder' which is available through the HNB portal helps users to locate the appropriate resources to answer their queries by employing a detailed, tree-like questionnaire. Furthermore, automated complex tool cascades ('tasks'), involving resources located on different servers, have been implemented, allowing users to perform comprehensive data analyses without the requirement of further manual intervention for data transfer and re-formatting. Currently, automated cascades for the analysis of regulatory DNA segments as well as for the prediction of protein functional properties are provided. AVAILABILITY: The HNB portal is available at http://www.hnbioinfo.de
Assuntos
Algoritmos , Biologia Computacional/métodos , Sistemas de Gerenciamento de Base de Dados , Armazenamento e Recuperação da Informação/métodos , Internet , Análise de Sequência de DNA/métodos , Análise de Sequência de Proteína/métodos , Interface Usuário-Computador , Biologia Computacional/organização & administração , Alemanha , Relações Interinstitucionais , SoftwareRESUMO
Cytoplasmic segments of the adult rat skeletal muscle sodium channel alpha-subunit (rSkM1) comprise a major portion (approximately 40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel's amino- and carboxy-termini and identify residues 1716-1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an alpha-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp1617.
Assuntos
Músculo Esquelético/química , Canais de Sódio/química , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Dicroísmo Circular , Primers do DNA/genética , Técnicas In Vitro , Proteínas Ligantes de Maltose , Oligopeptídeos , Peptídeos/química , Peptídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , Canais de Sódio/genética , Espectrometria de FluorescênciaRESUMO
To test sodium channel structural models, we defined the epitopes for nineteen independently cloned monoclonal antibodies previously generated against purified, detergent-solubilized, adult rat skeletal muscle sodium channel protein using channel proteolysis, synthetic peptides, and fusion proteins. All identified epitopes were continuous and unique to the skeletal muscle subtype alpha-subunit. Of the nineteen independent clones, seventeen had epitopes located either in the origin of the amino-terminus or in the interdomain 2-3 region while only two antibodies had epitopes located in the mid-portion of the interdomain 1-2 region. No immunogenic regions were identified on the alpha-subunit's extracellular regions, interdomain 3-4 segment, or carboxyl-terminus or on channel beta-subunits. While immune tolerance may explain the lack of immunogenicity of extracellular regions, the lack of immunogenicity of most of the channel's cytoplasmic mass may be due to segment inaccessibility from organization of these regions as globular domains, to insertion of parts of these regions into the membrane phase, or to interaction with other protein elements. The definition of monoclonal antibody epitopes allows us to reinterpret previously reported monoclonal antibody competition studies, providing independent support for our model of sodium channel cytoplasmic domain structure. In addition, these data suggest additional testable hypotheses concerning the interactions of the sodium channel amino- and carboxyl-termini with each other as well as with other protein elements.
