Date of Graduation

Spring 2016

Document Type

Honors Research Project

Degree Name

Bachelor of Science

Major

Chemistry - Biochemistry

Research Sponsor

Dr. Thomas Leeper

First Reader

Dr. Adam Smith

Second Reader

Dr. Claire Tessier

Abstract

This project investigates the cluster stability of an outer mitochondrial membrane protein mitoNEET under mutational stress and drug binding analysis. MitoNEET is a part of a class of proteins that coordinate an Fe-S molecule into its tertiary structure. This protein is believed to facilitate the oxidative capacity of the electron transport chain by reversible loss of its coordinated Fe-S molecule. The research of this paper uses site directed protein mutagenesis to selectively alter the three cysteine, one histidine cluster coordination into more common coordination patterns such as two cysteine, two histidine or four cysteine. The ultimate goal is to optimize conditions for future research and analysis by UV-Vis spectrometry. Research led to the conclusion that optimal conditions for kinetic analysis under acidic conditions were in a pH 5.5 20 mM citric acid reaction buffer with 100 mM NaCl and 5% DMSO. To the reaction buffer 100 mM protein and 100 mM drug were added. This reaction was carried out using the Shimadzu Spectra apparatus (Absorbance values at 459 nm) and the reaction cuvettes were temperature controlled by a water bath at 27°C to yield complete cluster loss within 6 hours.

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