Senior Honors Projects, 2010-current

Date of Award

Spring 2017

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Biology

Advisor(s)

Jonathon Monroe, Ph.D.

Christopher Berndsen, Ph.D.

Kimberly Slekar, Ph.D.

Abstract

Glutathionylation is a reversible post-translational modification of proteins involving the transfer of glutathione to the thiols of specific cysteine residues. While the mechanism behind glutathionylation is known, the specificity of cysteine glutathionylation is not understood. It is known, however, that the two main factors affecting the susceptibility to glutathionylation are the reactivity and accessibility of cysteines in proteins, which is determined by the microenvironment. Using β-amylases (BAMs) 1 and 3 from Arabidopsis thaliana, which have different sensitivities to nitrosoglutathione (GSNO), as a model, I attempted to provide insight into why some cysteines are glutathionylated by GSNO and others are not. Our lab found that GSNO inhibits BAM3 activity by glutathionylating Cys433 in vitro, yet BAM1 is unaffected by GSNO despite containing a cysteine at the same position. At a physiological level, the glutathionylation of BAM3 by GSNO may be important as BAM3 may be inhibited by a NO-induced modification under cold stress. Different microenvironments surrounding Cys433 in BAM1 and BAM3 could explain the different sensitivities these enzymes have to GSNO. I compared sequence alignments of BAM1 and BAM3 from a variety of flowering plants. If a position is important in making Cys433 in BAM3 and BAM1 sensitive and insensitive, respectively, to GSNO, then that position would likely be conserved within each BAM1 and BAM3 orthologous set but be different between the sets. After comparing sequence alignments and locating these positions in homology models, I hypothesized that H430, N432, and S434 might contribute to the sensitivity of Cys433 in BAM3 to GSNO, and that the corresponding amino acids in BAM1, D430, L432, and A434, might contribute to its insensitivity. To test this hypothesis, I made three BAM3 mutants with the amino acid substitution H430D, N432L, or S434A and the corresponding BAM1 mutants, D430H, L432N, and A434S. All of the BAM1 mutants were active but only the BAM3 mutant with the H430D substitution was active. I then treated the active mutants with GSNO and compared their sensitivity to the BAM3 and BAM1 controls. The BAM1-D430H and BAM1-A434S mutants were inhibited by 45% and 20%, respectively, by GSNO whereas the WT BAM1 control was insensitive to GSNO. The active BAM3 mutant, however, was just as sensitive to GSNO as the BAM3 control. Therefore, D430 and A434 may contribute to the insensitivity of Cys433 to glutathionylation by GSNO in BAM1. D430 may play a larger role in making Cys433 insensitive to GSNO in BAM1 than A434 because it is negatively charged, which could decrease the reactivity and/or the accessibility of Cys433.


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