Folding and assembly of dimeric human glutathione transferase A1-1

Glutathione transferases function as detoxification enzymes and ligand-bind ing proteins for many hydrophobic endogenous and xenobiotic compounds. The molecular mechanism of folding of urea-denatured homodimeric human glutathi one transferase A1-1 (hGSTA1-1) was investigated. The kinetics of change we re investigated using far-UV CD, Trp20 fluorescence, fluorescence-detected ANS binding, acrylamide quenching of Trp20 fluorescence, and catalytic reac tivation. The very early stages of refolding (millisecond time range) invol ve the formation of structured monomers with nativelike secondary structure and exposed hydrophobic surfaces that have a high binding capacity for the amphipathic dye ANS. Dimerization of the monomeric intermediates was detec ted using Trp fluorescence and occurs as fast and intermediate events. The intermediate event was distinguished from the fast event because it is limi ted by a preceding slow trans-to-cis isomerization reaction (optically sile nt in this study). At high concentrations of hFKBP, dimerization is not lim ited by the isomerization reaction, and only the fast event was detected. T he fast (tau = 200 ms) and intermediate (tau = 2.5 s) events show similar u rea-, temperature-, and ionic strength-dependent properties. The dimeric in termediate has a partially functional active site (similar to 20%). Final r eorganization to form the native tertiary and quaternary structures occurs during a slow, unimolecular, urea- and ionic strength-independent event. Du ring this slow event (tau = 250 s), structural rearrangements at the domain interface occur at/near Trp20 and result in burial of Trp20. The slow even t results in the regain of the fully functional dimer. The role of the C-te rminus helix 9 (residues 210-221) as a structural determinant for this fina l event is proposed.