Date of Graduation

Spring 5-31-2021

Document Type

Thesis

Degree Name

Master of Science in Chemistry

College/School

College of Arts and Sciences

Department/Program

Chemistry

First Advisor

Janet G. Yang, Ph.D.

Second Advisor

Lawrence Margerum, Ph.D.

Third Advisor

Osasere Evbuomwan, Ph.D.

Abstract

Chemical homeostasis is a baseline requirement for any cell to survive. ATP-binding cassette (ABC) transporters play a vital role in homeostasis by importing nutrients and exporting toxins against their concentration gradients by utilizing the energy of ATP hydrolysis. Malfunctioning ABC transporters cause a variety of health problems, including cystic fibrosis, Stargardt’s disease (vision loss), and the development of drug-resistant tumors. An important step in solving these medical issues is to first understand the structure and mechanism of ABC transporters. Various studies have made great strides in depicting the structure and details of different ABC transporters and their mechanisms, however, many of these details were discovered with transporters in highly artificial environments using X-ray crystallography. This project aims to further understand the mechanism of the E. coli methionine importer MetNI using functional studies.

A fluorescence anisotropy assay was developed as a functional study that would assess the dissociation constant between MetNI and its periplasmic binding protein MetQ. ATP-binding by MetNI was found to be a prerequisite for MetNI-Q complex formation. MetNI saw a slightly higher affinity for apo MetQ (Kd = 281 nM ± 36 nM) than L-Met bound MetQ (527 nM ± 107 nM). These similar binding affinities support the hypothesis that MetNI follows two different mechanisms originally proposed by Nguyen et. al.; one for the preferred L-Met substrate, and one for L-Met derivatives in situations of L-Met scarcity. Preliminary trials investigating the dissociation constant between the MetNI C2 domains and L-Met found a Kd dissociation constant between the MetNI C2 domains and L-Met of 484 nM. Moving forward, the ATP requirements for MetNI-Q complex formation will be investigated using MetNI transporters with nucleotide binding domain chimeras.


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