Date of Award
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Bachelor of Arts
Nicholas J. Kaplinsky
Small heat shock proteins (sHSP) are a diverse family of molecular chaperones which bind thermally denatured proteins and thereby prevent them from aggregating. This prevents damage to cells and facilitates their later refolding. Plant sHSP have been characterized biochemically, but their physiological importance is unclear because sHSP knock-out mutants have shown few phenotypes when subjected to heat stress, possibly because of redundancy. BOBBER1 (BOB) is 34.5 kDa Arabidopsis thaliana protein with homology to sHSPs that is induced by high temperatures and localizes to heat shock granules along with other sHSPs. bob1-3 mutants have thermotolerance defects and developmental irregularities, suggesting a novel role for BOB in the plant chaperone network. In this investigation, a recombinant 6xHis-tagged form of BOB protein was expressed in E. coli and purified. The ATP-independent chaperone activity of BOB was demonstrated; BOB was shown to reduce the thermal aggregation of the model substrates malate dehydrogenase and citrate synthase. Site-directed mutagenesis was used to create mutant alleles of BOB protein that were both computationally predicted to disrupt its structure and are available in plants. Truncated forms of the BOB N and C termini were also expressed and purified in an attempt to understand the roles of the protein domains in interaction with partially unfolded substrate. The N terminus did not reduce aggregation under any condition tested, while two different forms of the C termini showed reduced chaperone activity compared to full length protein. The point mutation found in thermotolerance defective bob1-3 mutants did not result in loss of chaperone activity, raising the possibility that the mutation disrupts interactions with specific substrate(s) or cochaperones which would not be reflected in our assay. Identification of these substrates may be required for an understanding of why BOB is necessary for normal development and thermotolerance.
Hoyer, Steen , '09, "In vitro characterization of the chaperone activity of the Arabidopsis non-canonical small heat shock protein BOBBER" (2009). Senior Theses, Projects, and Awards. 93.