ABSTRACT
Equilibrium unfolding, single- and double-jump kinetic studies were conducted to
determine the unfolding and refolding pathway of glutaredoxin 2. Structural
changes for wild-type glutaredoxin 2 were monitored by far-ultraviolet circular
dichroism and intrinsic tryptophan fluorescence for equilibrium unfolding and
intrinsic tryptophan fluorescence for single- and double-jump kinetics studies.
Glutaredoxin 2 possesses two tryptophan residues in domain 2. In order to
monitor changes in domain 1, cysteine 9 at the active site cysteines, situated in
domain 1, was labelled with an extrinsic fluorophore, AEDANS, and a mutant
was created (Y58W glutaredoxin 2). The AEDANS labelled protein displayed
decreased alpha-helical secondary structure and conformational stability. A high
degree of cooperativity and similar conformational stability was observed during
the two-state transition of the urea-induced equilibrium unfolding of both the
wild-type and Y58W glutaredoxin 2 proteins therefore Y58W glutaredoxin 2
could be used to assess structural changes in the local environment of domain 1
during unfolding and refolding. Two phases of unfolding, the fast and slow phase,
occurred for both the wild-type and Y58W proteins. The slow phase involves
structural rearrangements that expose small amounts of surface area while the fast
phase represents gross structural unfolding exposing large amounts of surface
area. The isomerization of the Val48-Pro49 peptide bond to the trans
conformation occurs during the slow phase and this isomerization is coupled to
conformational unfolding of the protein. The structural separation of these phases
could be represented by two structural units (unit x and unit y), these units do not
represent domain 1 and 2. The units could also result in parallel refolding
pathways with the folding of the x unit involving the fast and slow refolding
phases and the folding of the y unit of structure is represented by the medium
phase of refolding. The fast and slow phases are further separated as the fast
phase represents the gross structural folding of glutaredoxin 2 for species with the
Val48-Pro49 peptide bond in the native cis conformation. The development of the
slow phase after extended unfolding delay periods during double-jump refolding
studies, as well as the acceleration of the rate of the phase by the peptidyl prolyl
isomerase hFKBP-12 proved that the phase involves a proline peptide bond
iv
isomerization. This phase represents a slow isomerization coupled with
conformational folding similar to the slow unfolding phase. Complex unfolding
and refolding kinetics indicated the involvement of kinetic intermediates during
(un)folding.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/4845 |
| Date | 19 May 2008 |
| Creators | Gildenhuys, Samantha |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 8733326 bytes, application/pdf, application/pdf |
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