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1 Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea
2 Center for Biofunctional Molecules, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea
3 Department of Chemistry, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea
Reprint requests to: Kwan Yong Choi, Division of Molecular Life Sciences, Center for Biofunctional Molecules, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea; e-mail: kchoi{at}postech.ac.kr; fax: 82-54-279-2199.
Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida biotype B. Folding was reversible in secondary and tertiary structures as well as in activity. Equilibrium unfolding transition, as monitored by fluorescence and ellipticity measurements, could be modeled by a two-state mechanism without thermodynamically stable intermediates. Consistent with the two-state model, one dimensional (1D) NMR spectra and gel-filtration chromatography analysis did not show any evidence for a folded monomeric intermediate. Interestingly enough, Cys 81 located at the dimeric interface was modified 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 refolding process, as monitored by fluorescence change, could best be described by five kinetic phases, in which the second phase was a bimolecular step. Because <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolecular step. During the refolding process, negative ellipticity at 225 nm increased 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 
-helical structures before dimerization. Monitoring the enzyme activity during the refolding process could estimate the activity of the monomer that is not fully active. Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.
Keywords: Ketosteroid isomerase; folding; dimerization
Abbreviations: KSI, ketosteroid isomerase • PI, KSI of Pseudomonas putida biotype B • TI, KSI of Comamonas testosteroni • DTT, dithiothreitol • pSK(-), pBluescript SK(-) • EDTA, ethylenediaminetetraacetic acid • CD, circular dichroism • NMR, nuclear magnetic resonance • KD, dissociation constant • 
GUH2O, Gibbs free energy change for unfolding in the absence of urea and at 25°C • DTNB, 5,5'-dithio-bis(2-nitrobenzoate) • 5-AND, 5-androstene-3,17-dione
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