Characterization of the pH-dependent protein stability of 3α-hydroxysteroid dehydrogenase/carbonyl reductase by differential scanning fluorimetry

Abstract

The characterization of protein stability is essential for understanding the functions of proteins. Hydroxysteroid dehydrogenase is involved in the biosynthesis of steroid hormones and the detoxification of xenobiotic carbonyl compounds. However, the stability of hydroxysteroid dehydrogenases has not yet been characterized in detail. Here, we determined the changes in Gibbs free energy, enthalpy, entropy, and heat capacity of unfolding for 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) by varying the pH and urea concentration through differential scanning fluorimetry and presented pH-dependent protein stability as a function of temperature. 3α-HSD/CR shows the maximum stability of 30.79 kJ mol^-1 at 26.4°C, pH 7.6 and decreases to 7.74 kJ mol^-1 at 25.7°C, pH 4.5. The change of heat capacity of 30.25 ± 1.38 kJ mol^-1 K^-1 is obtained from the enthalpy of denaturation as a function of melting temperature at varied pH. Two proton uptakes are linked to protein unfolding from residues with differential pK_a of 4.0 and 6.5 in the native and denatured states, respectively. The large positive heat capacity change indicated that hydrophobic interactions played an important role in the folding of 3α-HSD/CR. These studies reveal the mechanism of protein unfolding in HSD and provide a convenient method to extract thermodynamic parameters for characterizing protein stability using differential scanning fluorimetry.

Keywords: Gibbs free energy; denaturant; differential scanning fluorimetry; enthalpy; entropy; heat capacity; protein stability curve; thermal unfolding.

FIGURE 1

pH dependence of thermal unfolding of 3α‐HSD/CR by DSF. (a) The thermal unfolding curve of 3α‐HSD/CR was sigmoidal at pH 4.5–9.7. The region of transition shifted to a higher temperature and appeared to be steeper at neutral pH than at acidic or alkaline pH values. The solid lines represent the fit of the data to Equation (1) to obtain T _m and ΔH _m , as listed in Table 1 and S1. All experiments were performed at least in triplicate at each pH value, and the representative fluorescence melting curves are presented. (b, c) pH‐dependences of T _m and ΔH _m , respectively. The T _m value increased as the pH increased from 4.5, reached a plateau of approximately 52°C at pH 7.6–8.7, then slightly decreased as the pH increased to 9.7. The ΔH _m value had a maximum value of 787 ± 17 kJ mol⁻¹ at pH 7.6 and decreased to 385 ± 46 and 372 ± 33 kJ mol⁻¹ at pH 4.5 and 9.7, respectively. Error bars represent the standard deviation of at least three independent measurements. (d) The linear relationship between ΔH _m and T _m at pH 4.5–7.6. The T _m and ΔH _m values at each pH are fitted to a linear equation, giving a slope of ΔC _p of 30.25 ± 1.38 kJ mol⁻¹ K⁻¹ and an intercept of −772.4 ± 63 kJ mol⁻¹ at 0°C. ΔH _U vanished at T _h of 25.5°C.

FIGURE 2

pH dependent ΔG _U of 3α‐HSD/CR. (a) ΔG _U /2.3RT as a function of pH at 25°C. The ΔG _U at pH 4.5–7.6 is listed in Table 1. The line shows that the maximum slope lies between a pH of 4.8 and 6.0, which is 2.0 ± 0.1, the corresponding number of proton uptakes (ΔQ) when the protein unfolds. (b) The ΔG _U values at pH 4.5–7.6 were analyzed by fitting the data to Equation (5), giving the pK_a of 4.0 ± 0.8 and 6.5 ± 0.2 of the residues of 3α‐HSD/CR in the folded and unfolded states, respectively.

FIGURE 3

Thermal unfolding curves of 3α‐HSD/CR in the presence of urea at pH 7.6 by DSF. (a) The fluorescence intensity of the thermal unfolding of 3α‐HSD/CR as a function of temperature in the presence of urea 0–2 M was monitored by DSF and showed a sigmoid curve. The thermal unfolding curves shifted toward lower temperatures as the urea concentration increased. The lines represent the fit of the data to Equation (1) to obtain T _m and ΔH _m at various urea concentrations. (b) Thermal unfolding curves of (a) were converted to a fraction of the unfolded protein at each temperature. At least three experiments were performed for each concentration of urea. The representative melting curves are shown. (c, d) T _m and ΔH _m values of 3α‐HSD/CR in the presence of 0–2 M urea at pH 7.6. Both T _m and ΔH _m decreased as the urea concentration increased. (e) The relationship between ΔH _m and T _m is linear in 0–1.1 M urea. The data were fitted to a straight line, giving a slope of apparent ΔC _p of 27.2 ± 1.7 kJ mol⁻¹ K⁻¹.

FIGURE 4

Free energy of the thermal unfolding of 3α‐HSD/CR induced by urea at various temperatures using DSF. (a) Thermal unfolding of 3α‐HSD/CR in 0–2 M urea using DSF. The fraction of unfolding of 3α‐HSD/CR between 0.05 and 0.95 in 0–2 M urea was converted to ΔG _U at various temperatures. ΔG _U in the presence of urea at temperatures between 41°C and 50°C are shown. The lines fit the ΔG _U at different concentrations of urea to Equation (7) at 41–50°C to obtain the ΔG _U (H₂O) and m values. The dashed line indicates that the ΔG _U is equal to 0, where 50% of the enzyme is in the denatured state. (b–d) Temperature dependence of ΔG _U (H₂O), m, and [urea]_0.5, respectively, for urea‐induced unfolding of 3α‐HSD/CR by DSF. Both ΔG _U (H₂O) and [Urea]_0.5 decreased as the temperature decreased, while the m value seemed to be independent of temperature in the range of 41–50°C within the error, giving an average of 2.7 ± 0.2 M.

FIGURE 5

Denaturant‐induced denaturation of 3α‐HSD/CR at various temperatures. (a) Relative intrinsic fluorescence intensity of 3α‐HSD/CR in the presence of urea at 15°C, 22°C, 25°C, and 30°C and GdmCl at 22°C, pH 7.6. The fluorescence intensity at 317 nm, measured at each concentration of denaturant, was normalized to that at zero concentration of denaturant. The lines represent the fit of the data to Equation (8). (b) The fraction of unfolding between 0.05 and 0.95 at various concentrations of urea or GdmCl was converted to ΔG _U . The lines represent the fitting of ΔG _U to the concentrations of urea or GdmCl at the indicated temperatures in Equation (7) to obtain the ΔG _U (H₂O) and m values. The intercepts of ΔG _U of urea‐ and GdmCl‐induced unfolding at 22°C were similar, indicating that the ΔG _U (H₂O) values obtained using different denaturants were similar.

FIGURE 6

(a) Two‐state model of protein denaturation induced by temperature or denaturants. The conformations of protein between the native state (N) and the denatured state (D) are in reversible equilibrium with the equilibrium constant K _U and ΔG _U which are affected by temperature or denaturants, such as acid, urea, and GdmCl. (b) Protein stability curves of 3α‐HSD/CR at pH 4.5–7.6. The stability of 3α‐HSD/CR decreased as the pH decreased from 7.6 to 4.5. Meanwhile, ΔG _U was obtained using different approaches at pH 7.6. The linear extrapolation method was used to evaluate ΔG _U through denaturant‐induced denaturation. The circle and diamond symbols represent the ΔG _U obtained from the urea‐ and GdmCl‐induced unfolding, respectively, by following the intrinsic fluorescence at the indicated temperatures. The triangles represent the ΔG _U near the melting temperature, which was determined by varying 0–2 M urea through DSF. These results are consistent with the ΔG _U in the stability curve, which was obtained by fitting the values of ΔH _m , T _m , and ΔC _p determined by DSF to Equation (3) at various temperatures (solid curve). The black dotted line indicates the ΔG _U of the maximum stability at T _s (Table 1) for the stability curve of 3α‐HSD/CR at the corresponding pH value, where ΔS _U is equal to zero and ΔG _U is purely enthalpic (ΔH _U (T)). (c) The pH‐dependence of protein stability shows a link between the equilibrium constants of proton binding (K _a‐N and K _a‐D) and conformational unfolding transitions (K _D‐N and K _D‐NH) in the two‐state model.