Identification of abscisic acid-binding proteins using a bioactive photoaffinity probe
Date
2009-05-15
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Doctoral
Abstract
This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin
based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.
Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of
equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA.
This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that
non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to
displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA
(phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has
suggested the selectivity of binding.
Further, Rubisco enzymatic activity in the absence of ABA was compared to that in
the presence of ABA at various concentrations. The results have clearly indicated the
effect of ABA on Rubisco’s enzymatic activity. This was reflected on the enzyme’s Km
values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate
(RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most
resembles allosteric inhibition.
The biological function of this newly discovered interaction is interpreted as
ABA’s ability to regulate plant growth during abiotic stress by its direct action on the
photosynthetic machinery - hypothesis often suggested in the literature.
Description
Keywords
Abscisic Acid (ABA), ABA-binding proteins, abiotic stress, Rubisco inhibition
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Chemistry
Program
Chemistry