Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B

Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida bi otype B. Folding was reversible in secondary and tertiary structures as wel l as in activity. Equilibrium unfolding transition, as monitored by fluores cence and ellipticity measurements, could be modeled by a two-state mechani sm without thermodynamically stable intermediates. Consistent with the two- state model, one dimensional (1D) NMR spectra and gel-filtration chromatogr aphy analysis did not show any evidence for a folded monomeric intermediate . Interestingly enough, Cys 81 located at the dimeric interface was modifie d by DTNB before unfolding. This inconsistent result might be explained by increased dynamic motion of the interface residues in the presence of urea to expose Cys 81 more frequently without the dimer dissociation. The refold ing process, as monitored by fluorescence change, could best be described b y five kinetic phases, in which the second phase was a bimolecular step. Be cause <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolec ular step. During the refolding process, negative ellipticity at 225 nm inc reased very fast within 80 msec to account for >80% of the total amplitude. This result suggests that the protein folds into a monomer containing most of the or-helical structures before dimerization. Monitoring the enzyme ac tivity during the refolding process could estimate the activity of the mono mer that is not fully active. Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.