RUI:MCB: Glutathionylation of beta-amylase3 in Arabidopsis thaliana

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This project will ultimately lead to an improved ability to predict how certain proteins can be modified under conditions of environmental stress. Such modifications may lead to changes in the protein's function that help organisms respond to altered conditions, including temperature change. The research is focused on a plant amylase, an enzyme that breaks down leaf starch and appears to play a role in cold stress survival. This enzyme is inhibited by a mechanism involving the reversible modification of certain amino acids that may occur during cold stress. This type of modification is poorly understood but is thought to be widespread in all organisms responding to environmental stress. The modified amino acids in the amylase have been identified and this project will characterize how these modifications affect enzyme activity and what it is that makes these amino acids sensitive to this specific modification. The research will be carried out at a predominantly undergraduate institution and involve at least 10 undergraduates, helping prepare them for graduate school, professional schools or other careers in science. The students will be involved in all aspects of the work and be able to present their results at regional and national conferences. In addition, the project will support a postdoctoral scholar as she gains experience in teaching and mentoring undergraduates in research, skills that are essential to her eventual goal of working at an undergraduate institution. Glutathionylation of proteins at specific cysteine residues is an important post-translational modification but little is known about what makes some cysteines sensitive to glutathionylation while others are insensitive. In addition, there are no examples in plants where the physiological effects of protein glutathionylation are understood. In this project the well-studied model plant Arabidopsis thaliana will be used to investigate two members of the β-amylase (BAM) gene family, one of which, BAM3, is inhibited by glutathionylation and the other, BAM1, is not. The two objectives of the project are: 1) to characterize the glutathionylation of BAM3 and the effects of this modification on catalytic activity using site directed mutagenesis, mass spectrometry and homology modeling, and 2) to identify key factors influencing the susceptibility of cysteines to glutathionylation. A conserved Cys residue was identified that is sensitive to glutathionylation in BAM3 but is insensitive in BAM1. Sequence alignments and homology modeling of BAM3 and BAM1 sequences across plants revealed a number of residues that differ between the two BAMs, but are conserved within each type and may influence the sensitivity to glutathionylation. A combination of computational modeling and protein mutagenesis will be used to characterize the factors affecting this glutathionylation event. Results from these experiments will lead to the development of a more accurate model for predicting the sensitivity of cysteine residues to glutathionylation in other proteins, and to a better understanding of the molecular mechanisms involved in stress acclimation.