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Date of Award

Spring 2012

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science


Biochemistry & Molecular Biol.

First Advisor

Dr. Ellis Bell


Malate dehydrogenase (MDH) is a well-studied member of the dehydrogenase family and one of the first multisubunit enzymes to have its structure solved using x-ray crystallography. However, many questions still remain about the mechanism and role of subunit interactions in MDH. In order to investigate subunit interactions and provide a quantitative framework for studying engineered interface mutants, a single cysteine residue was engineered to the outer edge, furthest from the interface and shown to be reactive. The reactive thiol group has been used to immobilize gMDH on the gold tip and stage of an atomic force microscope to begin pulling- type experiments. These experiments probe the effects of ligands on the stability of the dimer interface relative to the overall protein structure using analysis of the resultant force curves. In another approach to studying the effects of ligand binding, random labeling of the 2 engineered cysteines per dimer with Alexa Fluor 488/AF647 [Ro approximately 52Å] or AF 488/546 [Ro approximately 63Å] has been used to allow resonance energy transfer measurements between the two halves of the dimer. This method has been used to probe ligand-induced changes over long distances and effects on dimer stability.