An Affinity Probe for Isolation of Abscisic Acid-Binding Proteins

Abstract

An affinity probe has been developed for isolation of receptor proteins that bind the plant hormone abscisic acid (ABA). The structural features required for biological activity have been preserved, and the probe has been demonstrated to bind to known ABA-binding proteins.

Chemical biology of abscisic acid

“…Studies using biotin-labeled ABA possessing a biotin group at the 4 0 -position of ABA (Table 1) suggested the existence of plasma membrane receptors in barley aleurone and broad bean guard cells (Kitahata et al 2005a;Yamazaki et al 2003). In contrast, ABA with a biotinylated bicyclic structure (Table 1) bound to the ABA catabolic enzyme, CYP707A1 demonstrated in vitro the potency of biotin-labeled ABA for isolation of ABAbinding proteins (Nyangulu et al 2005). Caged ABA is an analogue with a substituted 1-(2-nitro)-phenylethylester in place of the carboxylic acid (Table 1).…”

Identifying the Cellular Targets of Bioactive Small Molecules with Activity-Based Probes

“…Biotinylated probes can be utilized in various ways in target identification strategies. They can be pre-complexed with a streptavidin resin to create a non-covalent affinity matrix [29][30][31][32][33][34][35][36], or incubated with live cells for desired time and then fished out of the solution with streptavidin resin after cell lysis [37][38][39][40]. The biotinylated probes and their attached proteins may be eluted from avidin resins by 8 M guanidineHCl, pH 1.5, or by boiling in a typical denaturing SDS-PAGE loading dye.…”

Studies of Abscisic Acid Perception Finally Flower

“…The structural requirements of ABA perception have been studied by testing the activity of the naturally occurring (1)-S form of ABA, its (2)-R enantiomer, and a variety of ABA metabolites and analogs in diverse ABA responses, including stomatal regulation, germination, and gene expression (Walton, 1983;Walker-Simmons et al, 1994Nyangulu et al, 2005). Several major conclusions have been derived from these studies: (1) most responses are specifically induced by the (1) form or some of its derivatives, but even (2) ABA is effective for some responses; (2) ABA metabolites, such as 8-hydroxy-ABA and phaseic acid that were initially considered inactivated derivatives of the hormone, still activate some responses; (3) different responses display different specificities; and (4) most changes to ABA decrease, but don't necessarily eliminate, its biological activity.…”

“…Although each of these studies demonstrated the bioactivity of the relevant ABA derivative, all of these derivatives were coupled through portions of the hormone previously identified as critical for activity and therefore might be compromised in interaction with (some) receptors. An alternate approach was recently described by Nyangulu et al (2005), who have developed a bioactive ABA derivative that displays all essential features of the hormone and is tethered to biotin via an additional ring structure. Although this affinity probe has been shown to bind anti-ABA antibodies and an ABA-metabolizing enzyme, it has not yet identified any candidate receptors.…”

“…Biochemical approaches with carefully designed affinity probes, such as that described by Nyangulu et al (2005), or follow-up on the ABA binding proteins that have been reported by Zhang et al (2002) and Yamazaki et al (2003) are likely paths to success.…”