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Date of Award

Spring 2008

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

First Advisor

Dr. J. Ellis Bell

Abstract

Intrinsic protein fluorescence, through tryptophan emission has long been a common tool to monitor protein structure and function. The use of this probe provides great insight into the tertiary structural integrity. Historically, these studies have often been limited only to enzymes that naturally contain tryptophan residues. However, with advances in site-directed mutagenesis techniques, the engineering of these probes into native enzymes with no native tryptophans has provided great insight into their structure and function, with minimal perturbation. Malate dehydrogenase is a highly conserved enzyme involved in metabolic processes throughout the cell. As a homo-dimer, malate dehydrogenase exhibits a complex mechanism of regulation hypothesized to be due to cross-subunit conformational communication between its two identical active sites. Native glyoxysomal malate dehydrogenase (gMDH) does not contain any tryptophan residues, making study of these conformational changes difficult to monitor directly. Here, we successfully engineer in fluorescent tryptophan residues into two locations, L229 and I319, one close and the other far from the subunit interface, respectively. According to Stern-Volmer quenching with I- and NADH, L229 is shown to be sensitive to ligand-induced conformational changes, signifying that these may occur at or near the subunit interface. To further investigate conformational reporting, we have designed, purified, and characterized a mutant that is catalytically dead and that contains the “probe” L229W mutation. Using gMDH’s unique ability to spontaneously refold after denaturation, in the future we hope to recombine an active subunit with this “dead-probe” subunit. This will allow for direct monitoring of local conformation events transmitted across the subunit interface.

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