The role of a conserved interdomain interaction in Escherichia coli glutaredoxin-2

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dc.contributor.author Parbhoo, Nishal
dc.date.accessioned 2010-08-26T12:21:33Z
dc.date.available 2010-08-26T12:21:33Z
dc.date.issued 2010-08-26
dc.identifier.uri http://hdl.handle.net/10539/8557
dc.description.abstract Domain interfaces play an important role in protein stability and folding. A major structural feature at the interdomain interface of the GST class of proteins is the conserved hydrophobic ‘lock-and-key’ motif. In a monomeric homologue of the GSTs, Grx2, the hydrophobic interdomain ‘lock-and-key’ motif is formed by insertion of the side-chain of methionine 17 (Met17) from domain 1 into a hydrophobic pocket in domain 2. This study evaluates the contribution of the Met17 residue to the stability of Glutaredoxin-2 (Grx2). Protein engineering techniques were employed to generate a Met17 to Alanine (M17A) mutant protein and comparative studies with wild-type and M17A Grx2 were performed. The spectral properties of M17A Grx2 monitored using far and near-ultraviolet circular dichroism and tryptophan fluorescence indicated no significant changes in secondary or tertiary structure in the native state. Conformational stability studies were performed to determine the contribution of the ‘lock-and-key’ motif to protein stability. Equilibrium unfolding studies, displayed significant impact on the conformational stability of the protein with a DDG(H2O) of 4 kcal.mol-1 as a result of the replacement of the Met17 residue with alanine. The co-operativity of unfolding is slightly decreased, with the mvalue being reduced by 0.3 kcal.mol-1.M-1 suggesting an intermediate formation. This intermediate becomes more prominent during equilibrium unfolding in the presence of ANS which showed an increase in intensity in the unfolding transition for M17A Grx2 but was absent for wild-type Grx2. The kinetics of unfolding of both Grx2 proteins are complex, both displaying two observable phases (fast and slow) which occur in parallel as confirmed by performing initial conditions test. 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 rate of the fast unfolding phase is increased for M17A Grx2, as the time constant decreased from 2.4s (wild-type) to 830ms, however there is negligible change in the rate of the slow phase. The increase in the unfolding rate of the fast phase is in agreement with the equilibrium studies which highlights the destabilisation as a result of the mutation. en_US
dc.language.iso en en_US
dc.title The role of a conserved interdomain interaction in Escherichia coli glutaredoxin-2 en_US
dc.type Thesis en_US


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