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Exploring the structure and function of uncoupling proteins: a biophysical approach

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dc.contributor.advisor Jelokhani-Niaraki, Masoud
dc.contributor.author Hoang, Tuan
dc.date.accessioned 2015-04-29T18:49:19Z
dc.date.available 2015-04-29T18:49:19Z
dc.date.copyright 2015-04
dc.date.created 2015-04-21
dc.date.issued 2015-04-29
dc.identifier.uri http://hdl.handle.net/10214/8774
dc.description.abstract Located in the mitochondrial inner membrane, uncoupling proteins (UCP) dissipate the proton electrochemical gradient across the membrane, resulting in the reduction of ATP synthesis. Abundantly expressed in the brown adipose tissue, UCP1 transports protons to the mitochondrial matrix and plays an important role in thermogenesis. Neuronal UCP homologs (UCP2, UCP4, and UCP5) may have crucial roles in the function and protection of the central nervous system. However, their structure and specific functions are poorly understood. The main goal of this study is to explore the structure and functional properties of mammalian UCPs, with a focus on the neuronal UCPs for which less information is available. Using recombinant protein expressions, all UCPs were produced in bacteria, purified, and reconstituted into liposomes for structural and functional studies. Conformations of UCPs were analyzed using circular dichroism and fluorescence spectroscopies. Proton and chloride transport assays were developed for reconstituted UCPs using the anion-sensitive fluorescent probe SPQ. Three main studies were done in this project. In the first study, the ion transport activity of neuronal UCPs was examined in vitro for the first time. The comparative conformational and ion transport study of neuronal UCPs provided fundamental information on their structure and function. In the second study, potential key amino acids, involved in ion transport of UCP2, were mutated. The effects of these residues on ion transport and protein conformation were examined in detail. Results of this study revealed the essential role of positively charged residues in TM2 in the protein’s chloride transport pathway. In the third study, optimized folding of recombinant UCP was reported using the pET26 vector containing a bacterial periplasmic targeting sequence. The incorporation of this leading sequence allowed UCPs to be expressed in the membrane of E. coli. All proteins exhibited helical structures in mild detergents and phospholipid bilayers and displayed proton transport function. Moreover, self-association of UCPs in lipid membranes was observed and characterized for the first time. This latter discovery can lead to new insights in the structure-function relationships of UCPs. Overall, these studies have important implications in understanding the structure and proton transport mechanism of UCPs in the mitochondria. en_US
dc.language.iso en en_US
dc.rights Attribution-NoDerivs 2.5 Canada *
dc.rights.uri http://creativecommons.org/licenses/by-nd/2.5/ca/ *
dc.subject membrane proteins en_US
dc.subject uncoupling proteins en_US
dc.subject UCP en_US
dc.subject reconstitution en_US
dc.subject liposomes en_US
dc.subject ion tranpsort en_US
dc.subject biophysics en_US
dc.subject biochemistry en_US
dc.title Exploring the structure and function of uncoupling proteins: a biophysical approach en_US
dc.type Thesis en_US
dc.degree.programme Biophysics en_US
dc.degree.name Doctor of Philosophy en_US
dc.degree.department Department of Molecular and Cellular Biology en_US
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Attribution-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NoDerivs 2.5 Canada