Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Dr. M. Jelokhani-Niaraki

Advisor Role

Supervisor/Professor

Abstract

Human Uncoupling Protein 2 (hUCP2) is one of five known human UCPs which are found in the inner mitochondrial membrane and have been shown to facilitate the translocation of protons from the intermembrane space to the mitochondrial matrix. The detailed physiological role of UCP2 proton transport, the mechanism by which it mediates this proton transport, as well as its structure has also yet to be elucidated. In order to help determine the topology of UCP2 embedded in the membrane as well as its mechanism of proton transport, the intrinsic fluorescence properties of the two tryptophan residues (Trp) present in its structure (W176 and W283) were used. To this end, three substitution mutants were created by site-directed mutagenesis using primer overlap-extension PCR; these were: W176Y, W283F and W176Y-W283F. The three mutant cDNAs then introduced into a membrane-targeted bacterial expression system from which they were then expressed in E. coli and purified via autoinduction and immobilized metal affinity chromatography (IMAC). Fluorescence and circular dichroism spectroscopy were then used to confirm that the mutants retained native-like conformations. Fluorescence quenching assays with acrylamide were performed to probe the topology of UCP2 embedded in the membrane via the differing accessibility of each Trp to the acrylamide quencher. The final stage of this project involved probing the effects of the Trp residues on the proton transport activity of UCP2 via a proton transport assay previously developed in our lab. Unfortunately, the presence of high background fluorescence, as seen in the W176Y-W283F fluorescence spectra with excitation at 295nm, either due to tryptophan containing-proteinaceous contaminants in the sample, or the presence of a deprotonated tyrosine residue (tyrosinate) in the folded structure of UCP2, has made it impossible to draw any definitive conclusions from the intrinsic fluorescence assays. The results of the proton transport assay for the W176Y-W283F Trp mutant and wt UCP2 indicate a role for the conserved UCP Trp residues in the proton transport activity of the protein based on a 26.7% reduction in the transport rate of the double mutant in comparison to the wt protein.

Convocation Year

2015

Convocation Season

Fall

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