When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.
Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.
The enzyme tRNA nucleotidyltransferase is of central importance in eukaryotic cells since it is required in multiple intracellular locations (nucleus, cytosol, mitochondria and plastids) for the production of mature tRNAs needed for protein synthesis. Therefore, tRNA nucleotidyltransferase serves as an excellent model protein to explore the complex roles that localization, activity, structure and stability play in defining protein function. I investigated how changes in the amino acid sequence of tRNA nucleotidyltransferase affect its structure, stability, activity and localization and may be linked, ultimately, to human disease.
Using Arabidopsis thaliana as a model organism, I was the first to show that amino acid substitutions located within a region of tRNA nucleotidyltransferase required for enzyme activity, but outside of the enzyme’s classical amino-terminal organellar targeting sequence, affect localization to plastids and mitochondria. These studies suggest a direct link between intracellular distribution and how tightly the protein has folded. Moreover, these data indicate that the same seven amino acid changes that result in altered stability also reduce tRNA binding and enzyme activity. To more clearly define the role that these amino acids have in enzyme structure and function and to determine whether changes in protein stability, substrate binding, or structure bring about altered localization, I have fine-structure mapped the region encompassing residues 399-420 and shown that a single amino acid substitution (K418E) can dramatically alter tRNA binding and enzyme activity without detectable effects on enzyme structure. Localization studies are now underway to determine whether the K418E substitution, or other single substitutions within this region, alters the intracellular localization of tRNA nucleotidyltransferase.
Using a second model organism, Saccharomyces cerevisiae, I have further explored the link between stability and activity in protein function. I showed that a single amino acid substitution (E189F) resulted both in a 5ºC decrease in thermal stability and 25-fold decrease in enzyme activity and caused a temperature-sensitive phenotype. Moreover, converting arginine 64 to tryptophan in the variant enzyme restored enzyme activity and suppressed the temperature-sensitive phenotype, but did not restore thermal stability. These data suggest that the temperature-sensitive phenotype is defined by the reduction in activity and not thermal stability. Moreover, these data were the first to suggest a role for conserved motif C in active site organization and tRNA nucleotidyltransferase function.
Finally, I applied the techniques and insight acquired from my studies of the Arabidopsis and yeast enzymes to explore how seven distinct amino-acid substitutions in human tRNA nucleotidyltransferase, all previously identified in patients suffering from sideroblastic anemia with B cell immunodeficiency, periodic fevers and development delay (SIFD), affect enzyme structure, stability and function in vitro. I showed that each of these mutations affects some combination of tRNA nucleotidyltransferase structure, activity, or stability. As expected, substitutions (T154I, M158V, L166S, R190I and I223T) within the conserved catalytic amino terminal region of the protein altered stability and/or activity but more interestingly two substitutions (I326T and K416E) in the less well conserved carboxy-terminal region of the enzyme altered catalytic efficiency, tRNA binding and quaternary structure.
These studies have shown that a protein’s function can be defined by a combination of some or all of its structure, stability, activity or localization.