90 research outputs found
Kinetische Untersuchungen zum Mechanismus der GTP-Hydrolyse durch humanes Guanylat-bindendes Protein 1
No full text
Molekularer Mechanismus der GTP-Hydrolyse durch das humane Guanylat-bindende Protein 1
No full textGuanylat-bindende Proteine (GBPs) sind Interferon-induzierte GTPasen mit einer einzigartigen katalytischen Aktivität: sie hydrolysieren GTP nicht nur zu GDP sondern weiter zu GMP. Als Mitglied der Dynamin-Superfamilie großer GTP-bindender Proteine ist das humane Guanylat-bindende Protein 1 außerdem in der Lage, seine enzymatische Aktivität, d.h. sowohl die GTP- als auch die GDP-Hydrolyse, durch Selbstassemblierung zu stimulieren.
In dieser Arbeit wurde der molekulare Mechanismus der Nukleotidhydrolyse durch hGBP1 mit Hilfe transienter kinetischer Methoden wie , sowie zeitaufgelöster Fourier-transformierter Infrarot-Spektroskopie untersucht. Die Geschwindigkeiten der einzelnen Reaktionsschritte wurden bestimmt und damit die Lebenszeiten der auftretenden Intermediate definiert. Basierend auf diesen Untersuchungen wird ein Reaktionsmodell für die hGBP1-katalysierte GTP-Hydrolyse vorgeschlagen, das auch die Selbstaktivierung der GTPase durch Dimerisierung beinhaltet
Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine–ParM
No full textThe development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It is shown in the present study that one of these biosensors, tetramethylrhodamine–ParM, can also monitor GDP. The biosensor can be used to measure micromolar concentrations of GDP on the background of millimolar concentrations of GTP. The fluorescence response of the biosensor is fast, the response time being &lt;0.2 s. Thus the biosensor allows real-time measurements of GTPase and GTP-dependent kinase reactions. Applications of the GDP biosensor are exemplified with two different GTPases, measuring the rates of GTP hydrolysis and nucleotide exchange.</jats:p
Nucleotide dependent cysteine reactivity of hGBP1 uncovers a domain movement during GTP hydrolysis
No full textAbstractAs a member of the dynamin superfamily human guanylate-binding protein 1 (hGBP1) binds and hydrolyses GTP thereby undergoing structural changes which lead to self-assembly of the protein. Here, we employ the reactivity of hGBP1 with a cysteine reactive compound in order to monitor structural changes imposed by GTP binding and hydrolysis. Positions of cysteine residues buried between the C-terminal domain of hGBP1 and the rest of the protein are identified which report a large change of accessibility by the compound after addition of GTP. Our results indicate that nucleotide hydrolysis induces a domain movement in hGBP1, which we suggest enables further assembly of the protein
G-tract RNA removes Polycomb repressive complex 2 from genes.
No full textPolycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes
The guanine cap of human guanylate-binding protein 1 is responsible for dimerization and self-activation of GTP hydrolysis
No full textA Fluorescent, Reagentless Biosensor for ADP Based on Tetramethylrhodamine-Labeled ParM
No full textFluorescence assays for ADP detection are of considerable current interest, both in basic research and in drug discovery, as they provide a generic method for measuring the activity of ATPases and kinases. The development of a novel fluorescent biosensor is described that is based on a tetramethylrhodamine-labeled, bacterial actin homologue, ParM. The design of the biosensor takes advantage of the large conformational change of ParM on ADP binding and the strong quenching of the tetramethylrhodamine fluorescence by stacking of the dye. ParM was labeled with two tetramethylrhodamines in close proximity, whereby the fluorophores are able to interact with each other. ADP binding alters the distance and relative orientation of the tetramethylrhodamines, which leads to a change in this stacking interaction and so in the fluorescence intensity. The final ADP biosensor shows ∼15-fold fluorescence increase in response to ADP binding. It has relatively weak affinity for ADP (<i>K</i><sub>d</sub> = 30 μM), enabling it to be used at substoichiometric concentrations relative to ADP, while reporting ADP concentration changes in a wide range around the <i>K</i><sub>d</sub> value, namely, submicromolar to tens of micromolar. The biosensor strongly discriminates against ATP (>100-fold), allowing ADP detection against a background of millimolar ATP. At 20 °C, the labeled ParM binds ADP with a rate constant of 9.5 × 10<sup>4</sup> M<sup>−1</sup> s<sup>−1</sup> and the complex dissociates at 2.9 s<sup>−1</sup>. Thus, the biosensor is suitable for real-time measurements, and its performance in such assays is demonstrated using a sugar kinase and a mammalian protein kinase
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