Document Type


Degree Name

Doctor of Philosophy (PhD)



Program Name/Specialization

Biological and Chemical Sciences


Faculty of Science

First Advisor

Dr. Masoud Jelokhani-Niaraki

Advisor Role

Assistance with planning and interpretation of the experiments and editing the document.

Second Advisor

Dr. Matthew D Smith

Advisor Role

Assistance with planning and interpretation of the experiments and editing the document.


Uncoupling proteins (UCPs) are regulated proton transporters of the mitochondrial inner membrane. UCP-mediated proton leak negatively impacts the rate of ATP synthesis. Despite the importance of their physiological role(s) in certain tissues, molecular aspects of UCPs’ structure-function relationships are not fully understood. The current study explores the tertiary and quaternary structure of UCP2, as well as its proton transport mechanism in lipid membranes. The proteins were expressed in the E. coli inner membrane, purified and reconstituted into liposomes. Proteins were characterized by semi-native SDS-PAGE. Circular dichroism spectroscopy (CD) and fluorescence quenching assays were utilized to study the conformation of proteins and evaluate their proton transport function, respectively. Molecular dynamics (MD) simulations were performed in parallel to investigate the protein structure and some details of its proton transport function at atomic and molecular levels. To study the structure of UCP2, the protein was purified both as monomers and as a mixture of monomers, dimers, and tetramers in detergent micelles. After reconstitution, UCP2 associated into functional tetramers regardless of the original oligomeric/molecular form that was used for reconstitution. Computational analysis suggested that the functional tetramer is in fact a pseudosymmetrical dimer of dimers capable of inducing asymmetry in the membrane structure. Tetrameric UCP2 had a biphasic conformation in which the orientation of cavities of monomers in each dimeric unit was similar but opposite to the monomers of the other dimeric unit. The differences in cavity orientations within a tetramer is consistent with an alternating access mechanism. Based on this mechanism, in order to transport their substrates, the cavity of mitochondrial carrier proteins opens to the matrix and intermembrane space alternately. Two salt-bridge networks, one close to the matrix (matrix network) and the other close to the intermembrane space (cytoplasmic network) have been previously suggested to play important roles in the alternating access mechanism. The UCP2 matrix network was modified in multiple ways using point mutations of K→Q, D→N, K→D and D→K (resulting in either partial or full disruption or inversion of the network by switching the two ends of salt-bridges) and the consequences of these modifications on proton transport and its inhibition by ATP were analyzed. Advisors Prof. Masoud Jelokhani Niaraki & Prof. Matthew D. Smith Afshan Ardalan Wilfrid Laurier University, 2020 ii The matrix network had a filtering role in proton transport of UCPs and evidence is provided that the network may consist of more salt-bridges than have been previously suggested (five vs. three). A biphasic proton transport model is proposed for tetrameric UCP2 in which the conformation of each functional dimer is either in one or the other mode of transport, acting as an on/off switch. ATP could interact with all positive residues of the matrix network (K38, K141, K239, R88, R185, R279) and thus interfere with the alternating open and close modes of the protein’s cavity. Overall, this thesis provides new insights into the tetrameric structure of UCP2 and the mechanism by which proton transport takes place and is regulated in the tetrameric protein.

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