Date of Completion

2015

Thesis Type

College of Arts and Science Honors

Department

Biochemistry

First Advisor

Christopher Francklyn, Ph.D

Second Advisor

Jose Madalengoitia

Keywords

Synthetase, Enzyme, Conformation, Inhibitor, Fluorescence, Aminoacylation

Abstract

Traditionally, threonyl-tRNA synthetase (ThrRS) has been studied for its canonical function of aminoacylation. Recently, however, the enzyme has been identified as a potent angiogenic factor. Both functions are influenced by a structural change within the enzyme. Upon binding of a specific inhibitor, borrelidin (BN), an alternative conformation is adopted, preventing angiogenic function. Mutant versions of ThrRS were generated using site-directed mutagenesis to mimic the BN-bound structure. The effects of these mutations on threonine-induced conformational changes were explored using steady state fluorescence. The greatest conformational impairment was seen with A460S and S488W which demonstrated 38% and 41% of the control response, respectively. Additionally, differential scanning fluorimetry was utilized to detail complex stabilization upon substrate binding. Specifically, the binding of threonine increased the melting temperature of the wild type enzyme by 9.82 °C, but had reduced effects on the other mutations, with S488W displaying the least (1.84 °C). Borrelidin binding was found to induce a biphasic melting trend for the wild type, S488W, and A460S constructs, in agreement with the proposed two-step binding mechanism by Ruan, et al. Future studies include: (i) investigating angiogenic potential of each construct using in vivo tube formation assay, and (ii) determining enzyme structure using X-ray crystallography.

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