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Publikationen - Molekulare Signalverarbeitung

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Hellmuth, A.; Calderón Villalobos, L. I. A. Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors (Lois, L. M.; Matthiesen, R. ). Meth. Mol. Biol 1450, 23-34, (2016) ISBN: 978-1-4939-3757-8 DOI: 10.1007/978-1-4939-3759-2_3

In receptor–ligand interactions, dissociation constants provide a key parameter for characterizing binding. Here, we describe filter-based radioligand binding assays at equilibrium, either varying ligand concentrations up to receptor saturation or outcompeting ligand from its receptor with increasing concentrations of ligand analogue. Using the auxin coreceptor system, we illustrate how to use a saturation binding assay to determine the apparent dissociation constant (K D ′ ) for the formation of a ternary TIR1–auxin–AUX/IAA complex. Also, we show how to determine the inhibitory constant (K i) for auxin binding by the coreceptor complex via a competition binding assay. These assays can be applied broadly to characterize a one-site binding reaction of a hormone to its receptor.
Publikation

Biondi, E.; Branciamore, S.; Fusi, L.; Gago, S.; Gallori, E. Catalytic activity of hammerhead ribozymes in a clay mineral environment: Implications for the RNA world. Gene 389, 10-18, (2007)

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Publikation

Calderón Villalobos, L.I.; Nill, C.; Marrocco, K.; Kretsch, T.; Schwechheimer, C. The evolutionarily conserved <em>Arabidopsis thaliana</em> F-box protein AtFBP7 is required for efficient translation during temperature stress Gene 392(1-2), 106-116, (2007)

In eukaryotes, E3 ubiquitin ligases (E3s) mediate the ubiquitylation of proteins that are destined for degradation by the ubiquitin-proteasome system. In SKP1/CDC53/F-box protein (SCF)-type E3 complexes, the interchangeable F-box protein confers specificity to the E3 ligase through direct physical interactions with the degradation substrate. The vast majority of the approximately 700 F-box proteins from the plant model organism Arabidopsis thaliana remain to be characterized. Here, we investigate the previously uncharacterized and evolutionarily conserved Arabidopsis F-box protein 7 (AtFBP7), which is encoded by a unique gene in Arabidopsis (At1g21760). Several apparent fbp7 loss-of-function alleles do not have an obvious phenotype. AtFBP7 is ubiquitously expressed and its expression is induced after cold and heat stress. When following up on a reported co-purification of the eukaryotic elongation factor-2 (eEF-2) with YLR097c, the apparent budding yeast orthologue of AtFBP7, we discovered a general defect in protein biosynthesis after cold and heat stress in fbp7 mutants. Thus, our findings suggest that AtFBP7 is required for protein synthesis during temperature stress.
Publikation

Schilling, S.; Stenzel, I.; von Bohlen, A.; Wermann, M.; Schulz, K.; Demuth, H.-U.; Wasternack, C. Isolation and characterization of the glutaminyl cyclases from <i>Solanum tuberosum</i> and <i>Arabidopsis thaliana</i>: implications for physiological functions Biol. Chem 388, 145-153, (2007)

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