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Abstract
The CLIC (Chloride Intracellular Channel) proteins are a family of highly conserved proteins that exist as both soluble proteins and integral membrane forms. There are six human members in the CLIC family (CLIC1-CLIC6). The CLIC proteins have been associated with many biological functions, from cell cycle regulation to the maintenance of cell structural integrity. It has been shown that CLIC proteins are capable of inserting into membranes and forming functional ion channels in the absence of other proteins in vitro. It has also been demonstrated that CLIC1 undergoes reversible redox controlled structural changes. However, the exact biological function and mechanism of CLIC proteins are still unclear.
Recent publications indicate that CLIC protein function may involve protein-protein interactions or S-nitrosylation. It has been shown that CLIC5-A interacts with the ERM protein ezrin, a protein that functions as a cross-linker between the plasma membrane and the actin cytoskeleton. In addition, it has been demonstrated that S-nitrosylation induces nuclear translocation of CLIC4.
In this thesis, the crystal structures of the FERM domain of ezrin (FERM-Ezrin), full-length ezrin, four CLIC1 Cys24 mutants (C24A, C24D, C24S and FLAG-C24S) and S-nitrosylated CLIC1 are presented. The structures of full-length ezrin (residues 1-297, 516-586) are missing the intermediate domain due to limited proteolysis that occurred without the addition of protease during crystallisation. I have shown that CLIC1 forms a weak non-covalent complex with ezrin in vitro. The complex formation involves the CLIC1 active site cysteine (Cys24) in its reduced state. I have demonstrated that CLIC1 and CLIC4 are S- nitrosylated in vitro where Cys59 and Cys89 are identified as the two S-nitrosylation sites in CLIC1 using protein crystallography.