Folding mechanism of Glutaredoxin 2
| dc.contributor.author | Gildenhuys, Samantha | |
| dc.date.accessioned | 2008-05-19T09:06:24Z | |
| dc.date.available | 2008-05-19T09:06:24Z | |
| dc.date.issued | 2008-05-19T09:06:24Z | |
| dc.description.abstract | 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. | en |
| dc.format.extent | 8733326 bytes | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10539/4845 | |
| dc.language.iso | en | en |
| dc.subject | protein folding | en |
| dc.subject | Thioredoxin | en |
| dc.subject | Glutathione-S-transferases | en |
| dc.subject | Glutaredoxin 2 | en |
| dc.subject | cis-trans Isomerization | en |
| dc.title | Folding mechanism of Glutaredoxin 2 | en |
| dc.type | Thesis | en |