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

Abstract

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 alpha-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.

Scheme 1.

KSI catalyzed isomerization reaction. The reaction is known to be stereospecific; the β proton at C4 of the substrate, 5-androstene-3,17-dione, is transferred to the catalytic base of the enzyme to generate the reaction intermediate in the middle, and then the same proton is transferred to the β side of C6 to generate the product, 4-androstene-3,17-dione.

Fig. 1.

Ribbon diagram of Pseudomonas putida KSI (Kim et al. 1997a) seen in a direction of twofold symmetry axis. The side chains responsible for fluorescence signal are displayed and labeled. Residues 1–131 and 201–331 refer to the two subunits, respectively. The program Molscript (Kraulis 1991) was used to draw the figure.

Fig. 2.

Reversibility of PI folding in secondary and tertiary structures. Fluorescence (A) and CD (B) spectra of native, unfolded, and refolded PI are displayed for comparison. The fluorescence spectra of the proteins were obtained after excitation at 285 nm. The protein concentration was 15 μM. The unfolded protein was prepared by incubating the protein at 7 M urea longer than 48 h. The refolded protein was prepared by diluting the unfolded protein to 0.2 M urea and incubating longer than 48 h. The experiments were performed at 25°C in the buffer containing 20 mM potassium phosphate at pH 7.0, 0.5 mM EDTA, and 1 mM DTT.

Fig. 3.

1D NMR spectra of PI at different urea concentrations. The protein was incubated in the presence of urea longer than 48 h for equilibration. The representative spectra obtained at 0, 3, 5.5, and 8 M urea are displayed. The refolded protein was prepared by dialyzing the denatured protein with the refolding buffer containing 20 mM potassium phosphate, 0.5 mM EDTA, and 3 mM DTT. The protein concentration was 500 μM for all of the cases. The spectra were obtained according to the protocol as described in Materials and Methods. The peaks labeled a, b, and c represent some of the side chain protons of Leu 70, Val 88, and Tyr 16/Tyr 57, respectively. DTT peaks are labeled d.

Fig. 4.

Equilibrium unfolding transition of PI. (A) The equilibrium unfolding transitions monitored by fluorescence and CD measurements are displayed. PI was incubated in the buffer containing 20 mM potassium phosphate at pH 7.0, 0.5 mM EDTA, 1 mM DTT, and urea at different concentrations. The fluorescence intensity at 320 nm (υ) and the maximum wavelength (×) were obtained after excitation at 285 nm, and the CD ellipticity was measured at 222 nm (O). The protein concentration was 15 μM. (B) Protein concentration dependence of the equilibrium unfolding transition. The equilibrium unfolding transition was analyzed by the fluorescence measurement at three different protein concentrations of 1, 5, and 25 μM. The Y-axis represents the fraction of unfolded protein. The data points were fitted to equation 2 to obtain the transition curve.

Fig. 5.

Size-exclusion chromatography analysis of PI. (A) Elution profiles are displayed for PI at different urea concentrations. The protein was incubated in the presence of urea at the indicated concentrations longer than 48 h. The incubated protein was then loaded onto the column equilibrated with the buffer containing 20 mM potassium phosphate, 0.5 mM EDTA, 10 mM β-mercaptoethanol at pH 7, and urea at the respective concentration. The flow rate was 0.4 mL/min and the protein peak was monitored by the absorbance at 280 nm. (B) Dependence of the peak elution time on the urea concentration. The elution time obtained at different urea concentrations was plotted against the urea concentration.

Fig. 6.

Chemical modification and equilibrium unfolding analysis of PI-C81. (A) The transition curves are compared between the chemical modification of the dimeric interface Cys 81 (×) and the equilibrium unfolding of PI-C81 (O). PI-C81 at 15 μM was incubated at different urea concentrations longer than 48 h. Just after the addition of DTNB, the absorbance at 412 nm was measured and plotted against urea. The equilibrium unfolding of PI-C81 was also analyzed by monitoring the protein fluorescence intensity at 320 nm after excitation at 285 nm. For comparison, the equilibrium unfolding transition curve for the wild-type PI is also displayed (ν). The Y-axis represents the fraction of modified or unfolded protein in the total protein. (B) Dependence of the equilibrium unfolding transition of PI-C81 on the protein concentration. The unfolding transition was analyzed by the fluorescence measurements at two different protein concentrations of 1 and 15 μM. The data points were fitted to equation 2 to obtain the transition curve.

Fig. 7.

PI refolding kinetics monitored by fluorescence measurement. The refolding was induced by diluting the denatured protein dissolved in 7 M urea to 0.64 M urea. The representative trace obtained for 5 μM PI is displayed. The fluorescence intensity passed through the 305-nm cutoff filter was monitored after the excitation at 285 nm. Five traces were accumulated to obtain the final data. The trace was best fitted by five exponential functions. Their relaxation times were 0.026, 0.26, 1.7, 12.5, and 770 sec, respectively, in the subsequent order of the kinetic phases.

Fig. 8.

Dependence of the second refolding phase on the protein concentration. (A) Representative traces of PI refolding monitored by fluorescence change are displayed at the final protein concentrations of 0.4, 1, 5, and 15 μM. The Y-axis represents the normalized fluorescence change with taking the fluorescence maximum and minimum as 1 and 0, respectively. (B) Dependence of the rate constant of the second phase on the protein concentration. The second-order rate constant was determined to be ∼8 × 10⁵ M⁻¹sec⁻¹ in the range of low protein concentrations.

Fig. 9.

Catalytic effect of Cyclophilin A on the fourth and fifth refolding phases. The refolding trace of PI was obtained by monitoring the fluorescence change in the presence of Cyclophilin A. The PI concentration was 3 μM, and two different concentrations, 1 and 3 μM, of Cyclophilin A were tried. The rate constants for the fourth and fifth phases were plotted against Cyclophilin A concentration. The ratio of k/k₀ was calculated from the refolding rates in the presence (k) and the absence (k₀) of Cyclophilin A.

Fig. 10.

PI refolding kinetics monitored by ellipticity change at 225 nm. The ellipticity change was observed until 6000 sec after a manual mixing. The unfolded protein was induced to refold by diluting the urea concentration from 7 to 0.64 M. The protein concentration was 5 μM. The Y-axis represents the mean residue molar ellipticity. The inset represents the kinetic trace in the range of 0–4 sec obtained by use of the Bio-Logic stopped-flow system. Ten traces were accumulated to obtain the final data. The initial ellipticity value (ν) for the unfolded protein is indicated.

Fig. 11.

Dependence of half-time for the activity recovery from denatured states on the protein concentration. The time required to reach the maximal slope of the reactivation rate was taken as the half-time. The half-time was plotted against the protein concentration (ν). The activity was assayed by using 5-AND as a substrate by monitoring the absorbance change at 248 nm. The refolding buffer was 34 mM potassium phosphate at pH 7.0, 1 mM EDTA, and 1 mM DTT. For comparison, the half-times were also displayed for TI (O) (Kim et al. 2000b).