Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Dr. Matthew Smith

Advisor Role

Primary Supervisor

Second Advisor

Dr. Masoud Jelokhani-Niaraki

Advisor Role

Co-Supervisor

Abstract

A proton gradient across the inner mitochondrial membrane (IMM) is required for the synthesis of adenosine triphosphate (ATP) via ATP synthase. In other words, generation of the proton gradient is coupled to the synthesis of ATP. Interestingly, uncoupling proteins (UCPs), also located in the IMM, dissipate the proton gradient, meaning that the gradient is not efficiently used for the production of ATP.1 The mechanism of proton transport activity of UCPs has been hypothesized and widely debated for the past 20 years to also reduce oxidative stress by reducing the production of reactive oxygen species (ROS).2 The majority of research on plant UCPs (also referred to as Plant Uncoupling Mitochondrial Proteins, or PUMPs) has focused on the UCP1 protein of Arabidopsis thaliana (AtUCP1). There is, however, relatively little known about the structure and function of the UCP1 protein, which is similarly true for its mammalian homologues. The research conducted herein contributes to filling this knowledge gap by expressing and purifying functional AtUCP1 and AtUCP2 proteins, then using biophysical approaches to study their structure and function to compare it in parallel to hUCP2. The results show both proteins expressed had high α-helical content in both mild detergent and liposome environments tested, and the observations suggest a quaternary structure for both proteins to exist as a mixture of monomers and dimers for the UCPs. This work investigates the impact of different membrane lipid components with a primary focus on phosphatidylcholine (PC), and the impact of cardiolipin (CL), on the structure and function of both AtUCP1and AtUCP2. Both have been reconstituted into liposomes that resemble the lipid composition of the IMM for analyses of influence on the structure. A preliminary functional analysis has been conducted that indicated under these conditions that AtUCP1 and AtUCP2 were both capable of transporting protons with AtUCP2 having a lower efflux rate of the two, but both were similarly inhibited by ATP. This ii functional analysis is compared to recent literature that suggests that AtUCP2 is directed to the Golgi and is not a true uncoupling protein like AtUCP1.

Convocation Year

2025

Convocation Season

Spring

Download

Share

COinS