Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Dr. Masoud Jelokhani-Niaraki

Advisor Role

Principal Investigator

Abstract

ABSTRACT

Uncoupling protein 2 (UCP2) is one of five UCP homologues found in the inner mitochondrial membrane that transports protons from the intermembrane space to the mitochondrial matrix. In turn, the proton motive force is dissipated and less ATP is produced in the mitochondria. UCP2 is proposed to influence insulin secretion in type II diabetes, and decrease the amount of reactive oxygen species produced in the mitochondria, however the detailed mechanism of ion (proton and anions) transport in UCP2 and other UCP homologues are not fully understood. Sequence alignment analysis performed on proteins in the mitochondrial carrier family (MCF) including UCPs, identified a matrix network of positively and negatively charged residues that were proposed to form salt bridges and mediate substrate translocation through the proteins. In this study, the positively charged lysine residues in the matrix network were investigated for their influence on the proton transport and nucleotide binding activity of UCP2. For this reason, four UCP2 mutants: K38Q, K141Q, K239Q, and K38Q/K239Q (double mutant) and native proteins were expressed in bacterial membranes. After which the conformation of the purified proteins was analyzed with far-UV circular dichroism (CD). Finally, a fluorescence-based assay was used to study the proton transport and nucleotide binding properties of the proteins. The overall conformations of the proteins were α-helical but the shifts in negative ellipticity at 208 nm and 222 nm observed for the mutants inferred a change in the helical packing of these proteins compared to the wild type. In addition, the mutant proteins had proton transport rates that were 35% (K38Q and K239Q) and 65% (K141Q and double mutant) less than the native UCP2. In the presence of ATP, the proton transport rates of the mutant proteins decreased by 3-6% except for K38Q that had a 38% decrease in proton transport activity. In summary, these results revealed that the positively charged lysine residues in the matrix network could participate in a salt bridge interaction that regulates the degree of helical packing, the ion transport activity and nucleotide binding properties in UCP2.

Convocation Year

2016

Convocation Season

Fall

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