CLIC1 is an intracellular membrane protein that is unusual in that it can exist
in both a soluble and an integral membrane form. The manner in which this
protein inserts into membranes is unknown although it is proposed to undergo a
change in structure whereby it initially experiences a degree of unfolding and
then refolds into its new membrane-bound conformation. This study focuses on
the characterisation of CLIC1 in terms of its secondary, tertiary and quaternary
structure, the determination of its conformational stability at equilibrium and
the establishment of its unfolding kinetics, all under conditions of varying pH,
polarity, redox conditions, temperature and ionic strength. CLIC1 was found to
be most stable at pH 7.0 / 20oC. The unfolding process is two-state and
cooperative, producing a DG(H2O) of ~10 kcal/mol and a m-value of ~2
kcal/mol per molar urea. A decrease in pH to 5.5 or an increase in temperature
to 37oC resulted in the stabilisation of an equilibrium intermediate species
under mild denaturing conditions and a destabilisation of the native state. This
was further evidenced by an increase in the rate of unfolding of CLIC1 from
the native state to the denatured state under these conditions. A state with
similar properties to the intermediate species was detected in the absence of
urea at pH 5.5 / 37oC and under non-reducing conditions at both pH 7.0 / 20oC
and pH 5.5 / 20oC. The intermediate species is more hydrophobic than either
the native or denatured state; it is stabilised by salts, has a reduced secondary
structure, increased flexibility and a buried Trp35 relative to the native state.
The rate of formation of the intermediate species is a slow process which may
involve an oligomerisation step. The results from this study provide an
interpretation for the structure and mechanism of CLIC1 pore formation in vivo
by comparing the effects of the environment on the structure and stability of
the protein.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/4853 |
| Date | 20 May 2008 |
| Creators | McIntyre, Sylvia |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 25634715 bytes, application/pdf, application/pdf |
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