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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.

Kopycki, J.; Schmidt, J.; Abel, S.; Grubb, D. Chemoenzymatic synthesis of diverse thiohydroximates from gluconsinolate-utilizing enymes from <i> Helix pomatia</i> and <i> Caldicellulosiruptor saccharolyticus</i> Biotechnol. Lett 33, 1039-1046, (2011) DOI: 10.1007/s10529-011-0530-y

Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.
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