Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Masoud Jelokhani-Niaraki

Advisor Role

Main supervisor

Second Advisor

Matthew D. Smith

Advisor Role

Co-supervisor

Abstract

Located in the inner mitochondrial membrane of brown adipose tissue, uncoupling protein -1 (UCP1) dissipates the proton electrochemical gradient, causing reduction in the rate of ATP synthesis, and generates heat by non-shivering thermogenesis. Three other UCP homologs (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have potential roles in the function and protection of the central nervous system (CNS). Up to date, structural information for UCPs still remains limited. Extensive biochemical studies on UCP2 have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in proteoliposome systems have been performed on UCP4 and UCP5. Thus, the goals of this study are to gain further information on the conformations and functional properties of neuronal UCPs reconstituted in liposomes. The emphasis is on UCP4 and UCP5 and their comparison to UCP2. Recombinant versions of all neuronal UCPs were successfully expressed, purified and reconstituted in azolectin liposomes (with and without the supplement of 2.5 mol% cardiolipin). UCP2 and UCP4 showed a high α-helical content in liposomes, while UCP5 conformation was less helical, with conformations exhibiting β-sheet characteristics. Ion transport assays (proton and chloride) for reconstituted neuronal UCPs were successfully developed using anion-sensitive fluorescent probes. All neuronal UCPs displayed proton transport across the membrane with characteristics similar to the archetypical protein UCP1, which is activated by fatty acids and inhibited by purine nucleotides. Chloride anion transport is also shown for UCP2 and UCP4. In addition, it was observed that the mitochondrial lipid cardiolipin (CL) induced drastic changes in conformation and ion transport of reconstituted UCPs. A hypothesized interaction mechanism of UCPs and CL was drawn from experimental results and molecular modelling. Overall, this study has provided a detailed picture of the conformation and ion transport properties of neuronal UCPs in liposomes, and also emphasizes the crucial role of cardiolipin in UCP structure and function.

Convocation Year

2011

Convocation Season

Fall

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