Ph.D, Faculty of Science, University of the Witwatersrand, 2011 / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/11336 |
| Date | 23 February 2012 |
| Creators | Legg-E'Silva, Derryn Audrey |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
Page generated in 0.0115 seconds