Thomas Bekman

Date of Award

2017

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Chemistry

Abstract

The goal of this thesis was to analyze structural features in proteins and how they relate to thermal stability. To this end, I attempted to generate three mutants of C. elegans cytoplasmic malate dehydrogenase (cMDH) based on the known structures of other malate dehydrogenases. Malate dehydrogenase is an ideal model because it is highly conserved and present in organisms that live in a wide range of temperatures. Organisms that live at higher temperatures require more stable enzymes to carry out their basic life processes. Hence, their enzymes tend to have more structural features that confer stability than their cooler environment counterparts. More stable features include a greater number of intersubunit interactions, more salt bridge formation, and lower internal cavity volume. PCR site-directed mutagenesis was used to create three cMDH-encoding plasmids containing stability altering mutations. Out of the three, one (the S67D mutant) was successfully expressed and isolated. Kinetics assays were then performed on the mutant and wild type enzymes to compare their substrate binding, specific activity, and stability. The S67D mutant bound oxaloacetate less tightly, had a lower specific activity, and was slightly more stable. The first two changes were unexpected, while the increase in stability was in line with hypothesized results.

Download

Share

COinS