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Date of Award
Restricted Thesis: Campus only access
Bachelor of Science
Biochemistry & Molecular Biol.
Dr. Ellis Bell
Malate Dehydrogenase (MDH) is a homodimeric oxidoreductase that catalyzes the NAD+/NADH‐dependent reversible oxidation of L‐malate into oxaloacetate.1 The enzyme is involved in the Kreb’s and glyoxylate cycles, as well as the malate‐aspartate shuttle.2 Different isoforms of MDH have been identified including cytosolic, mitochondrial, glyoxysomal, chloroplast, and bacterial, and these isoforms are located in various subcellular areas with specificity for NAD+ or NADP+.3 Each subunit of MDH has two structurally and functionally diverse domains: an NAD+‐binding domain in the amino‐terminal domain and a carboxy‐ terminal domain with substrate binding sites with an active site between these two domains.1 Citrate has been shown to regulate mitochondrial and glyoxysomal MDH and pH is expected to regulate cytosolic, glyoxysomal, and mitochondrial MDH, so citrate and pH are effectively allosteric regulators. The aim of this project is to study the linkage between pH and citrate regulation of malate dehydrogenase and protein stability and folding cooperativity. The hypothesis of this research is that there is a linkage between protein folding cooperativity and allosteric regulation, and therefore, citrate and pH will affect protein folding cooperativity. The cooperativity factor reflects the tertiary structure of the protein and its destabilization as interactions in this structure begin to break, leading to the coordinated unfolding of the protein, and hence may be related to conformational flexibility thought to be essential for allosteric regulation.
Circular dichroism spectroscopy and thermal melts using the circular dichroism instrument have been performed to gain insight into the secondary and tertiary structures of MDH and to show the unfolding cooperativity via the steepness of the unfolding transition (throughout the paper circular dichroism and thermal melts will be referred to as CD and TM, respectively). Citrate binds only one of the two active sites of MDH, therefore resulting in non‐competitive inhibition, resulting in a unique mode of allosteric interactions where a heterotropic ligand (citrate) elicits its effects by binding at the active site of one subunit in the dimer.2 At pH 6.0, clear effects on secondary structure have been detected using CD. TM using the CD spectrometer at 222 nm with data fitting via a four parameter sigmoidal equation to give both midpoint value and a cooperativity parameter indicate effects on the unfolding cooperativity, as well as the overall stability.
Comparison between results at pH 6.0 through pH 8.0, show a change in secondary structure at pH 6.0 based on the CD data and results at pH 6.0 only show regulation by citrate of MDH, suggesting effects of pH and citrate on folding cooperativity. While there is little effect at higher pH levels, the structure is different at pH 6.0, and citrate seems to have some effect at this pH. Analysis of the data indicates that low pH values have an effect on protein structure although the pH has no significant effect on overall stability. At low pH values, citrate seems to enhance stability. In relation to the hypothesis that protein folding cooperativity and allosteric regulation are linked, and citrate and pH will affect protein folding cooperativity, the data show a trend that supports this hypothesis at pH 6.0, but at higher pH values, there is no supporting evidence.
Yanta, Helen, "The linkage between citrate and pH regulation of Malate Dehydrogenase and protein folding" (2012). Honors Theses. 69.