The role of electrostatic interactions in the stability and structural integrity of human CLIC1

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dc.contributor.author Legg-E'Silva, Derryn Audrey
dc.date.accessioned 2012-02-23T08:02:34Z
dc.date.available 2012-02-23T08:02:34Z
dc.date.issued 2012-02-23
dc.identifier.uri http://hdl.handle.net/10539/11336
dc.description Ph.D, Faculty of Science, University of the Witwatersrand, 2011 en_US
dc.description.abstract Chloride intracellular channel proteins (CLICs) are able to exist in a soluble or membrane-bound state. The mechanism by which the transition between the two states takes place is yet to be elucidated. It is proposed that structural rearrangements of the N-terminal domain take place when CLICs encounter the lower pH environment of the membrane surface (pH 5.5). This prompts the CLICs to form a soluble membrane-ready state prior to pore formation and membrane transversion. Since the insertion of CLIC1 into membranes occurs at low pH, perhaps protonation and electrostatic effects of key conserved residues at the domain interface situated within the transmembrane region bring about the structural changes necessary for this transition. Structural and sequence alignments revealed that a conserved salt-bridge interaction between conserved residues on helices 1 and 3 of the N-terminal domain is present at the domain interface of CLICs. Therefore, this interaction was proposed to play an important role in maintaining the structural integrity and conformational stability of the N-terminal domain. This hypothesis was tested by mutating conserved CLIC1 residues Arg29 and salt-bridge partner Glu81 to methionine, thus removing the salt-bridge interaction. The conformational stabilities of each mutant at pH 7 (cytosol) and pH 5.5 (membrane surface) in the absence of membranes was then measured and compared to that of the wild type protein. The mutations did not impact upon the structural integrity of the protein. However, removal of the salt-bridge and hydrogen bonding interactions caused a loss in the cooperativity of unfolding from the native to unfolded state that resulted in the formation of an intermediate species. The intermediate species are less stable than the intermediate species of wild type CLIC1 at pH 5.5. Nevertheless, the properties (secondary and tertiary structure, ANS binding and cooperative unfolding (N ↔ U)) of the intermediate species are the same for all mutants and wild type protein. It can be concluded that the salt-bridge and more importantly hydrogen bonding interactions between helices 1 and 3 stabilise the Nterminal domain of CLIC1. It can be hypothesised that in the absence of membranes under acidic conditions, such as those at the surface of the membrane, protonation of acidic amino acid residues at the domain interface cause destabilisation of the Nterminal domain. This causes a reduction in the activation energy barrier for the conversion of soluble CLIC1 to its membrane-insertion conformation. en_US
dc.language.iso en en_US
dc.subject Membrane proteins en_US
dc.subject Biochemistry en_US
dc.title The role of electrostatic interactions in the stability and structural integrity of human CLIC1 en_US
dc.type Thesis en_US


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