Domain-domain interface packing at conserved Trp-20 in class alpha glutathione transferase impacts on protein stability.
The folding and assembly of the dimeric glutathione transferases (GST) involves the association of two structurally distinct domains per subunit. A prominent and conserved domain-domain interaction in class alpha GSTs is formed by the packing of the indole side chain of Trp-20 from domain I into a hydrophobic pocket in domain II. Stability studies have shown that partial dissociation of the domains near Trp-20 occurs as an initial fast event during the unfolding kinetics of human GSTA1-1 (Wallace et al., Biochemistry 37 (1998) 5320-5328; Wallace et al., Biochem. J. 336 (1998) 413-418). The contribution of Trp-20 toward stabilising the domain-domain interface was investigated by mutating it to either a phenylalanine (W20F) or alanine (W20A) and determining the functionality (catalysis and non-substrate ligand binding) and stability (thermal- and urea-induced denaturation) of the mutant proteins. The replacement of Trp-20 did not impact on the protein's gross structural properties. Functionally, the W20F was non-disruptive, whereas the cavity-creating W20A mutation was. Both mutants destabilised the native state with W20A exerting the greatest effect. Reduced m-values as well as the protein concentration dependence of the urea unfolding transitions for W20F GSTA1-1 suggest the presence of a dimeric intermediate at equilibrium that is not observed with wild-type protein. Unfolding kinetics monitored by stopped-flow tyrosine fluorescence was mono-exponential and corresponded to the global unfolding of the protein during which the dimeric intermediate unfolds to two unfolded monomers. The similar unfolding kinetics data for wild-type and W20F A1-1 indicates that the global unfolding event was not affected by amino acid replacement. We propose that the packing interactions at the conserved Trp-20 plays an important role in stabilising the intrasubunit domain I-domain II interface of class alpha GSTs.
Wallace
L A
LA
Protein Structure-Function Research Programme, Department of Molecular and Cell Biology, University of the Witwatersrand, 2050, Johannesburg, South Africa.
Burke
J
J
Dirr
H W
HW
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry*; D014508:Urea
D014364:Tryptophan; D014508:Urea; D005982:Glutathione Transferase