The role of a conserved interdomain interaction in Escherichia coli glutaredoxin-2
Date
2010-08-26
Authors
Parbhoo, Nishal
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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.