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.

1. INTRODUCTION

Protein stability is essential for understanding protein folding, protein structure–function relationship, and the application of protein preparations in drug discovery and development (Deller et al., 2016; Goldenzweig & Fleishman, ²⁰¹⁸). In addition, the conformational stability and dynamics of enzymes contribute to enzyme catalysis, which is involved in metabolism (Amaral et al., 2017; Callender & Dyer, ²⁰¹⁵; Richard et al., ²⁰¹⁸; Venkitakrishnan et al., ²⁰⁰⁴). In contrast, conformational disorders or misfolding of proteins can lead to protein aggregation, which is associated with various diseases including Alzheimer's disease, Huntington's disease, and cystic fibrosis. These diseases are characterized by the accumulation of abnormal protein aggregates in tissues and organs, leading to cellular dysfunction and damage (Chaudhuri & Paul, 2006; Chiti & Dobson, ²⁰¹⁷).

Protein stability is related to the Gibbs free energy of denaturation (ΔG _U ), which is the difference between the Gibbs free energy of the denatured and native states. Upon protein unfolding, the energy required to break the interactions between the residues in the folded state is counterbalanced by the hydration of the residues and conformational randomness in the unfolded state. These interactions, including hydrogen bonds, polar interactions, van der Waals forces, hydrophobic interactions, and salt bridges, contribute differently to protein stability (Nick Pace et al., 2014). The hydrophobic effect causes the formation of structured water around nonpolar groups with high heat capacity and low entropy in an unfolded state, thereby favoring the formation of a folded state by sequestering the nonpolar groups into the protein core from the solvent. The hydrophobic effect and loss of conformational entropy are the two main factors determining the stability of the folded state of a protein (Dill, 1990; Pace et al., ²⁰¹¹; Robertson & Murphy, ¹⁹⁹⁷). The average protein was marginally stable, with a ΔG _U of 20–42 kJ mol⁻¹ at physiological temperatures (Goldenzweig & Fleishman, 2018).

ΔG _U is determined by studying protein denaturation under a small range of conditions, such as temperature or the concentration of the chemical denaturants of acid, urea, or guanidine hydrochloride (GdmCl). The transition from the native state to the denatured state is highly cooperative and can be analyzed using a two‐state model. The Gibbs free energy is composed of enthalpy and entropy. Both the enthalpy of denaturation (ΔH _U ) and entropy of denaturation (ΔS _U ) are temperature‐dependent and depend on the change in heat capacity (ΔC _p ) between the native and denatured states. The value of ΔC _p is positive and large for protein denaturation, suggesting an important role for hydrophobic interactions in the exposure of buried nonpolar side chains to the solvent (Baldwin, 1986). Therefore, thermodynamic parameters ΔG _U , ΔH _U , ΔS _U , and ΔC _p are required to characterize the energetics of protein stability as a function of temperature. Differential scanning calorimetry was used to directly measure the excess heat change during unfolding as the temperature increased, giving ΔC _p and the enthalpy of denaturation ΔH _m at temperature T _m , where half of the protein molecules were denatured (Bruylants et al., 2005; Johnson, ²⁰¹³; Wen et al., ²⁰¹²). The values of ΔH _m and T _m were also determined by changing the equilibrium constants with temperature during thermal denaturation experiments. The changes in the unfolded and folded states with increasing temperature were monitored using a spectrophotometer. However, the analysis of the melting curves in a narrow temperature range at the transition is not sufficiently accurate to directly determine ΔCp. Alternatively, ΔC _p can be obtained from the slope of the linear relationship between ΔH _m and T _m , which is determined by thermal unfolding at various pH values (Becktel & Schellman, 1987; Privalov, ¹⁹⁷⁹). ΔG _U is also affected by the addition of denaturants such as urea or GdmCl. The Gibbs free energy for unfolding in the absence of denaturant, ΔG _U (H₂O), can be estimated by linear extrapolation of ΔG _U (D) determined in the transition region of unfolding at various denaturant concentrations (Pace, 1986). The protein stability curve, a plot of ΔG _U as a function of temperature, can then be obtained by applying the experimental determinations of ΔH _m , T _m , and ΔC _p to the Gibbs–Helmholtz equation to characterize the energetics of protein stability (Becktel & Schellman, 1987).

Differential scanning fluorimetry (DSF) has emerged as a rapid biophysical method for evaluating protein stability in relation to ligands, inhibitors, and pH. It is useful in the initial screening of proteins for small‐molecule interactions and in early stage drug discovery (Bergsdorf & Wright, 2018; Li & Zhang, ²⁰²¹; Niesen et al., ²⁰⁰⁷). The DSF monitors the thermal unfolding of proteins in the presence of a fluorescent dye using an RT‐PCR machine. As temperature increases, the proportion of unfolded proteins increases, leading to the hydrophobic parts of the protein being exposed to bind with a dye, thereby increasing the intensity of fluorescence (Gao et al., 2020). A sigmoidal curve was typically observed, indicating cooperative protein unfolding. A two‐state model of the transition from the native state to the denatured state was applied to analyze the curve, yielding T _m and ΔH _m (Matulis et al., 2005). DSF can be applied as a powerful high‐throughput tool to determine thermodynamic parameters of protein unfolding (Huynh & Partch, 2015; Wright et al., ²⁰¹⁷).

Hydroxysteroid dehydrogenase plays a role in the biosynthesis of steroid hormones and regulates the intracellular levels of biologically active steroid hormones by interconverting carbonyl and hydroxyl groups to bind their receptors (Lukacik et al., 2006; Hilborn et al., ²⁰¹⁷). 3α‐hydroxysteroid dehydrogenase/carbonyl reductase (3α‐HSD/CR) from Comamonas testosteroni catalyzes the oxidoreduction of androsterone with NAD⁺ to form androstanedione and NADH. 3α‐HSD/CR initiates steroid degradation by oxidizing a hydroxyl group at the C3 position to form a ketone group (Maser et al., 2001; Xiong & Maser, ²⁰⁰¹). 3α‐HSD/CR is a homodimer with an unresolved flexible substrate‐binding loop at T188‐K208 (Grimm et al., 2000). Loop closure upon substrate binding is important for better interactions with the transition state and for facilitating catalysis. However, conformational changes in the enzyme to the closed form can result in an entropic penalty (Hwang et al., 2017; Hwang et al., ²⁰¹⁹). To gain insights into the function of enzymes, it is crucial to determine their conformational stability (Shoichet et al., 1995). Enzymes undergo conformational changes and motions for their function, such as exclusion of solvents, binding with ligands, recruitment of essential residues for catalysis, stabilization and prevention of the loss of reactive intermediates, facilitation of transition‐state formation, and release of products (Johnson & Holyoak, 2010; Schramm et al., ²⁰⁰⁸). However, enzymes in a folded state with higher flexibility, which increases their conformational entropy, lead to a reduction in entropy gain during denaturation. This results in an increase in the free energy required for the unfolding and stabilization of the protein. For example, flexible P450 CYP119 was found to have higher thermal stability owing to the change in entropy during thermal unfolding compared with mesophilic CYP101A1 (Liu et al., 2018).

Structural stability has been studied by thermodynamic analysis of reversible protein denaturation induced by denaturants, such as pH, urea, and GdmCl. Furthermore, DSF is a rapid and high‐throughput method for obtaining T _m values for thermal unfolding of proteins. To characterize the conformational stability of 3α‐HSD/CR, we studied the energetics of protein stability as a function of temperature at various pH values and urea concentrations using intrinsic tryptophan fluorescence and DSF measurements. We determined the T _m , ΔG _U , ΔH _U , ΔS _U , and ΔC _p of thermal unfolding and constructed the protein stability curve of 3α‐HSD/CR, which showed the most stable with ΔG _U of 30.79 kJ mol⁻¹ at pH 7.6, 26.4°C and decreased significantly as the pH decreased. Furthermore, we developed an alternative method for the determination of ΔG _U (H₂O) from the pretransition and transition regions of urea‐induced protein that unfolds at various temperatures near the T _m through DSF. This study provides a more detailed picture of the stability and denaturation of 3α‐HSD/CR and a methodology to determine the energetics of protein stability.

2. RESULTS

2.1. Determine T _m , ΔH _m , and ΔC _p for thermal unfolding of 3α‐HSD/CR at various pHs by DSF

The stability of 3α‐HSD/CR as a function of temperature at various pH values was studied using DSF. Thermal unfolding of 2 μM 3α‐HSD/CR with 5× SYPRO Orange was performed by RT‐PCR at a heating rate of 0.5°C min⁻¹ from 25°C to 85°C. Because the fluorescence intensity of SYPRO Orange was significantly quenched at a pH below 4.5 (Figure S1), pH‐dependent thermal unfolding of 3α‐HSD/CR was carried out in 100 mM buffer at pH 4.5–9.7. The fluorescence intensity of the protein denaturation curve, as a function of temperature at different pH values, was sigmoidal (Figure 1a). Meanwhile, the thermodynamic parameters ΔH _m and T _m obtained from thermal unfolding by DSF were independent of both the concentration of 3α‐HSD/CR from 1 to 12 μM at pH 7.1 and the heating scan rate between 0.5°C and 2.0°C min⁻¹ at pH 4.5–7.6 by DSF (Figure S2). These results are consistent with a two‐state model of the native and denatured states. The pH profile of the thermal unfolding of 3α‐HSD/CR shows that the transition region of the curves appears at higher temperatures and steeper at neutral pH and shifts toward lower temperatures with smoother transitions to different extents at acidic and alkaline pH values. Each dataset of fluorescence intensity, including a linear baseline for the initial and final states as a function of temperature, was fitted to the two‐state model using Equation (1) to obtain T _m and ΔH _m , giving the temperature at which 50% of the protein was denatured and the change in enthalpy, respectively. The values of T _m and ΔH _m are given in Table 1 and S1 at pH 4.5–7.6 and pH 7.8–9.7, respectively. Plots of the pH‐dependent T _m and ΔH _m values are shown in Figure 1b, c, respectively. The pH‐dependent ΔH _m of 3α‐HSD/CR had a maximum value of 787 ± 17 kJ mol⁻¹ at pH 7.6 and decreases significantly in acidic and alkaline pHs with 385 ± 46 and 372 ± 33 kJ mol⁻¹ at pH 4.5 and 9.7, respectively. The T _m value increased from 38.3 ± 0.8°C to 51.6 ± 0.1°C as the pH increased from 4.5 to 7.6, reached a plateau at pH 7.6–8.7, and then decreased slightly to 50.5 ± 0.4°C as the pH increased further to 9.7. The pH‐dependent thermal stability showed that 3α‐HSD/CR was most stable at neutral pH but less stable under acidic conditions than under alkaline conditions.

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.

TABLE 1.

Thermodynamic analysis of 3α‐HSD/CR using protein stability curves.

pH	T m (°C) a	ΔH m (kJ Mol−1) a	ΔH m,calc (kJ Mol−1) b	T s (°C) b	ΔG U (T s ) (kJ Mol−1) b	ΔG U (25°C) (kJ Mol−1) b
4.5	38.3 ± 0.8	385 ± 46	385	25.7	7.74	7.70
4.7	38.8 ± 0.4	410 ± 17	402	25.7	8.41	8.37
4.8	41.4 ± 0.4	439 ± 38	477	25.8	11.84	11.80
5.1	44.2 ± 0.4	598 ± 13	565	25.9	16.23	16.19
5.6	46.8 ± 0.5	649 ± 33	644	26.1	20.88	20.79
6.0	49.3 ± 0.3	715 ± 17	720	26.3	25.90	25.77
6.5	50.5 ± 0.2	753 ± 25	757	26.3	28.41	28.33
7.1	51.2 ± 0.2	766 ± 33	774	26.4	29.92	29.79
7.6	51.6 ± 0.1	787 ± 17	787	26.4	30.79	30.67

^a T _m and ΔH _m were obtained by fitting the thermal unfolding curves obtained from DSF experiments using Equation (1). The errors represent the standard deviations of at least three measurements.

^b The thermodynamic parameters ΔH _m,calc, T _s , ΔG _U (T _s ), and ΔG _U (25°C) in the protein stability curves of 3α‐HSD/CR (Figure 6) were calculated using an appropriate equation. ΔH _m,calc was calculated using the equation $∆H_{m,\text{calc}}=30.25\times T_m-772.4$, using the measured T _m . The maximum stability occurred in the T _s , which was calculated using the equation of $T_s=T_m/\exp \left[∆H_{m,\text{calc}}/\left(T_m∆C_p\right)\right]$. ΔG _U (T _s ) and ΔG _U (25°C) were obtained by applying the values of T _m and ΔH _m,calc in Table 1, and ΔC _p of 30.25 kJ mol⁻¹ K⁻¹ to Equation (3) at T _s and 25°C, respectively.

The melting curve analyzed using Equation (1) provides the thermodynamic parameters of ΔH _m and T _m at various pH values. ΔC _p was determined from the linear relationship between ΔH _m and T _m based on Kirchhoff's law (Equation (2)). The dependence of ΔH _m on T _m from the pH‐dependent thermal unfolding of 3α‐HSD/CR appeared to be linear at pH 4.5–7.6, but no linear dependence at pH 7.8–9.7. Therefore, we selected the data at pH 4.5–7.6 to fit a linear equation to determine ΔC _p of the denaturation of 3α‐HSD/CR, as shown in Figure 1d. The slope gives a value 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 for the thermal unfolding of 3α‐HSD/CR. This result allowed us to analyze the ΔH _U of 3α‐HSD/CR at each temperature (T) from the equation $∆H_U=30.25\times T-772.4$ and yields ΔH _U  = zero kJ mol⁻¹ at T _h  = 25.5°C.

2.2. Determination of the number of proton uptakes during the unfolding of 3α‐HSD/CR at pH 4.5–7.6

The pH‐dependent protein stability can be affected by differences in the number of protons bound to native and denatured proteins at a given pH (Pace et al., 2009). The ΔG _U values calculated using Equation (3) at pH 4.5–7.6, 25°C are shown in Table 1 and Figure 2. The slope of the curve reflects the proton uptake (or release) during protein unfolding at pH (Oliveberg et al., 1995). The maximum slope of the curve was between pH 4.8 and 6.0. We determined the number of proton uptakes (ΔQ) during protein denaturation at pH 4.8–6.0 by fitting the data to Equation (4), giving a slope of 2.0 ± 0.1 (Figure 2a). The results indicated the uptake of two protons when 3α‐HSD/CR was unfolded, which could be caused by the differential ionization of residues between the folded and unfolded states. We assumed that these two protons originated from the same residue of each monomer because of homodimeric 3α‐HSD/CR in solution (Hwang et al., 2009). We determined the ionized state of the residue that participated in pH‐dependent protein stability at pH 4.5–7.6. The ionization constant of the residue in the folded and unfolded states was estimated by fitting the data to Equation (5). The best fit on the ionization constant of a residue is pK_a of 4.0 ± 0.8 and 6.5 ± 0.2 in the folded and unfolded states of 3α‐HSD/CR, respectively (Figure 2b).

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.

2.3. Effect of urea on T _m and ΔH _m for the thermal unfolding of 3α‐HSD/CR determined using DSF

The stability of 3α‐HSD/CR was further studied in the presence of urea at pH 7.6 by DSF. The fluorescence intensity and its conversion to fractional unfolding of 3α‐HSD/CR as a function of temperature in the presence of urea are shown in Figure 3. Thermal unfolding of 3α‐HSD/CR in 0–2 M urea showed a sigmoid curve, consistent with a two‐state model. Furthermore, thermal unfolding of 3α‐HSD/CR is sensitive to low concentrations of urea. The melting curves shifted toward a lower temperature, along with a gradually decreasing steepness of the transition, as the concentration of urea increased. The T _m and ΔH _m values in the presence of 0–2 M urea were obtained by fitting the data to Equation (1) and are listed in Table S2. The T _m is 51.6 ± 0.1°C in the absence of urea and decreases to 39.6 ± 0.1°C in the presence of 2 M urea. Plots of T _m and ΔH _m as functions of urea concentration are shown in Figure 3c, d, respectively. Both T _m and ΔH _m decreased as the urea concentration increased. Meanwhile, the relationship between ΔH _m and T _m determined in the presence of 0–1.1 M urea was linear. The apparent ΔC _p value was evaluated from the linear relationship between ΔH _m and T _m obtained in the presence of various concentrations of urea. Data are fitted to a linear equation, giving an apparent ΔC _p of 27.2 ± 1.7 kJ mol⁻¹ K⁻¹ at pH 7.6 (Figure 3e), which agreed with that obtained by thermal unfolding by varying the pH from 4.5 to 7.6.

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.

2.4. An alternative determination of ΔG _U (H₂O) in low concentrations of urea at various temperatures using DSF

We observed that the thermal unfolding of 3α‐HSD/CR was affected by low urea concentrations. Therefore, we analyzed the thermal unfolding of protein molecules in 0–2 M urea using DSF to determine ΔG _U (H₂O) at various temperatures. We converted the fraction of protein unfolding induced by urea from the data in the transition region of the unfolding curve between 0.05 and 0.95 in Figure 3b into ΔG _U (D) by Equation (6). The melting temperatures of 3α‐HSD/CR were 51.6°C and 39.6°C in 0 and 2 M urea, respectively. Hence, most of the protein is denatured when temperature is >51.6°C, and in its native form when the temperature is <39.6°C. To ensure that the data were analyzed for most of the proteins that occurred in the transition region induced by urea, we studied the linear relationship between ΔG _U (D) and urea concentrations from 41°C to 50°C by fitting Equation (7) (Figure 4a), giving ΔG _U (H₂O) as the intercept in the absence of urea, the slope of the m value, and [urea]_0.5 (Table 2). The midpoint of the denaturation that occurred at [urea]_0.5, where ΔG _U (D) was zero, was obtained from the ratio of ΔG _U (H₂O) to m. The dependence of ΔG _U (H₂O), m, and [urea]_0.5 as a function of temperature is shown in Figure 4b‐d. ΔG _U (H₂O) is 19.66 ± 0.54 kJ mol⁻¹ at 41°C and decreases to 3.18 ± 0.21 kJ mol⁻¹ as the temperature increases to 50°C. The m values with errors showed little temperature dependence. The average value of m is 11.3 ± 0.8 kJ mol⁻¹ between 41°C and 50°C. The [urea]_0.5 is 1.81 M at 41°C decreases to 0.25 M at 50°C. This study combines thermal‐ and urea‐induced protein unfolding by DSF and provides an alternative approach for determining the ΔG _U (H₂O) of unfolding at temperatures in the pretransition and transition regions induced by low concentrations of denaturants.

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.

TABLE 2.

Temperature dependence of the urea‐induced unfolding of 3α‐HSD/CR ^{a , b}

T (°C)	ΔG U (H2O) (kJ Mol−1)	m (kJ M−1)	[urea]0.5 (M)
15	29.46 ± 1.26	11.25 ± 0.50	2.61
22	30.75 ± 2.51	11.92 ± 0.96	2.57
25	29.29 ± 1.34	10.96 ± 0.50	2.67
30	25.98 ± 2.55	11.05 ± 1.09	2.35
41	19.66 ± 0.54	10.88 ± 0.33	1.81
42	17.82 ± 0.71	10.79 ± 0.50	1.65
43	16.02 ± 0.75	10.84 ± 0.54	1.48
44	14.43 ± 0.63	10.96 ± 0.50	1.32
45	12.93 ± 0.46	11.09 ± 0.38	1.17
46	10.88 ± 0.38	11.05 ± 0.42	0.98
47	8.79 ± 0.33	10.96 ± 0.42	0.80
48	7.07 ± 0.21	11.51 ± 0.29	0.61
49	5.44 ± 0.25	12.38 ± 0.42	0.44
50	3.18 ± 0.21	12.64 ± 0.46	0.25

^a Urea‐induced unfolding of 3α‐HSD/CR at temperatures of 15, 22, 25, and 30°C was determined using a spectrofluorometer.

^b Parameters from 41°C to 50°C were determined by DSF in the presence of 0–2 M urea.

2.5. Denaturant‐induced protein denaturation at various temperatures determined by a spectrofluorometer

The conformational stability of 3α‐HSD/CR was further analyzed by following intrinsic tryptophan fluorescence to monitor the structural changes induced by urea and GdmCl at various temperatures using a spectrofluorometer. 3α‐HSD/CR has a single Trp176 in the amino acid sequence. Upon exciting 3α‐HSD/CR at 295 nm, the emission spectrum of the native enzyme showed a λ_max of 330 nm in the absence of urea, which gradually shifted to 355 nm (a denatured state) as the concentration of either urea or GdmCl increased. The fluorescence emission spectra of the enzyme in the presence of urea at 15°C, 22°C, 25°C, and 30°C, and GdmCl at 22°C, pH 7.6 are shown in Figure S3. By comparing the fluorescence spectrum of 3α‐HSD/CR in the native state with that of the denatured state, the maximum difference in fluorescence intensity was observed at 317 nm. Therefore, the progress of denaturation of the fluorescence intensity at 317 nm was measured as a function of urea concentration, as shown in Figure 5a. The G_U(H₂O) and m values at different temperatures were obtained by fitting the change in intrinsic fluorescence intensity at 317 nm as a function of urea concentration to Equation (8). The data for urea‐induced unfolding are shown in Table 2. The values of ΔG _U (H₂O), m value, and [D]_0.5 are 31.0 ± 2.5 kJ mol⁻¹, 12.1 ± 0.8 kJ M⁻¹ and 2.6 M for the urea‐induced unfolding, and 28.0 ± 1.7 kJ mol⁻¹, 31.0 ± 1.7 kJ M⁻¹ and 0.9 M for the GdmCl‐induced unfolding at 22°C, pH 7.6, respectively. Alternatively, ΔG _U (H₂O) can be determined by extrapolating the linear relationship between ΔG _U (D) and the concentration of urea or GdmCl. ΔG _U (D) in the presence of urea or GdmCl was obtained from the equilibrium constant between the denatured and native states by fitting the observed fluorescence intensity to Equation (6). The fraction of unfolding between 0.05 and 0.95 in the presence of urea or GdmCl was converted to ΔG _U at various temperatures. A plot of ΔG _U (D) versus the concentrations of urea and GdmCl is shown in Figure 5b. The data were fitted to a straight line using the linear extrapolation method (Pace, 1986). The values of ΔG _U (H₂O), m, and [D]_0.5 for urea‐induced unfolding are 28.5 ± 2.5 kJ mol⁻¹, 11.3 ± 0.8 kJ M⁻¹ and 2.6 M at pH 7.6, respectively. The values of ΔG _U (H₂O), m, and [D]_0.5 for GdmCl‐induced unfolding are 27.2 ± 0.8 kJ mol⁻¹, 29.7 ± 0.8 kJ M⁻¹, and 0.9 M at pH 7.6, respectively. ΔG _U (H₂O), obtained using different denaturants, showed similar results.

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.

3. DISCUSSION

The denaturation of 3α‐HSD/CR is highly cooperative with the transition from a native state to a denatured state occurring over a small range of denaturation conditions by heat or chemical denaturants such as acid, urea, or GdmCl. We assumed a reversible two‐state model for the thermal denaturation of 3α‐HSD/CR in the DSF experiments, based on the following observations: 1. Thermal unfolding of 3α‐HSD/CR in the presence and absence of denaturants detected by DSF was a sigmoid curve, suggesting a two‐state model of native and denatured states with a negligible population of intermediates. 2. The thermodynamic parameters ΔH _m and T _m obtained from the data fitted to the two‐state model through the Gibbs–Helmholtz equation were repeatable and independent of the concentration of 3α‐HSD/CR and the heating rate of DSF. A kinetic mechanism for the reversible conversion of the native state to the denatured state, followed by an irreversible step, could cause a scan‐rate dependence on the thermodynamic parameters ΔH _m and T _m (Lepock et al., 1992). We studied the thermal unfolding of 3α‐HSD/CR at various concentrations (1–12 μM) using DSF, yielding identical values of ΔH _m and T _m within experimental errors. 3. We determined ΔG _U with different probes analyzed using the two‐state model, showing a similar result. The ΔG _U values obtained by temperature‐ and urea‐induced denaturation were similar. The unfolding of 3α‐HSD/CR was induced by heat and denaturants such as urea, GdmCl, and protons, and its progress was monitored by the change in fluorescence intensity detected by fluorometry or RT‐PCR. These probes yielded nearly identical ΔG _U. Therefore, we performed thermal unfolding of 3α‐HSD/CR using DSF to extract the thermal parameters ΔH _U , ΔS _U , ΔC _p , and ΔG _U for the characterization of protein stability based on a two‐state model of the native and denatured states as shown in Figure 6a.

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.

We determined the T _m and ΔH _m values for thermal unfolding of 3α‐HSD/CR using DSF. As temperature increases with DSF, the equilibrium between the denatured state and the native state changes. The dependence of the equilibrium constant on temperature allows us to deduce reliable values of ΔH _m and T _m along with the value of ΔC _p , which is usually not accurately determined. Therefore, ΔC _p is usually determined by perturbing the T _m of the protein through a change in pH, giving a linear relationship between ΔH _m and T _m to obtain the slope of this line, which reflects the ΔC _p of the denatured and native states. Similarly, T _m can be perturbed by the addition of urea to enhance protein unfolding. It has been shown that ΔC _p has a similar value in the presence of a low concentration of urea (Liu et al., 2001; Nicholson & Scholtz, ¹⁹⁹⁶). We determined the ΔC _p from a linear relationship between ΔH _m and T _m perturbed by pH and urea on thermal unfolding of 3α‐HSD/CR through DSF, giving a similar result of 30.1 and 27.2 kJ mol⁻¹ K⁻¹, respectively. The large positive value of ΔC _p for the denaturation of 3α‐HSD/CR indicates that the hydrophobic effect plays a significant role in the folding of 3α‐HSD/CR, and that a significant hydrophobic area is exposed to the solvent during unfolding. The exposure of buried nonpolar side chains to aqueous solutions can cause water to restructure, forming a clathrate structure surrounding exposed nonpolar residues. This ordered water shell requires more energy to disrupt interactions, leading to an increase in the heat capacity of the protein upon denaturation (Baldwin, 1986; Becktel & Schellman, ¹⁹⁸⁷).

The stability curves of 3α‐HSD/CR at different pH values were obtained by fitting the values of ΔC _p of 30.25 kJ mol⁻¹ K⁻¹, T _m , and ΔH(T _m ) from Table 1 to Equation (3), as shown in Figure 6b. However, the protein stability curve obtained from the equation based on the experimental values of T _m , H _m , and ΔC _p displayed significant extrapolation from the data near T _m . To validate the robustness of the protein stability curve of 3α‐HSD/CR as a function of temperature, we determined ΔG _U (H₂O) by denaturant‐induced denaturation and compared it with the values of the extrapolated stability curves obtained from pH‐dependent thermal unfolding data using DSF. ΔG _U (H₂O) values of 31.0 ± 2.5 and 30.2 ± 1.7 kJ mol⁻¹ were determined for urea‐ and GdmCl‐induced denaturation using the linear extrapolation method (Pace, 1986) at 22°C, pH 7.6, respectively (Table 2). These results were consistent with the ΔG _U of 29.79 kJ mol⁻¹ obtained from the analysis of the protein stability curve of 3α‐HSD/CR. Similarly, ΔG _U (H₂O) obtained from urea‐induced denaturation at 15°C, 25°C, and 30°C also lies near the protein stability curve shown in Figure 6b. We further determined ΔG _U (H₂O) using the linear extrapolation method from ΔG _U (D) obtained from urea‐induced denaturation at temperatures near the T _m . However, determination of ΔG _U (H₂O) using denaturant‐induced unfolding at higher temperatures is complicated. A shift in equilibrium favors the formation of a denatured protein as temperature increases, making it difficult to determine the baseline of the intrinsic fluorescence of the native state owing to the presence of an unknown amount of denatured protein. Furthermore, a long extrapolation of the transition region of protein unfolding induced by urea can cause errors in obtaining the ΔG _U (H₂O). Therefore, we developed an alternative methodology to determine ΔG _U (H₂O) from the pretransition and transition regions of protein unfolding induced by urea in the range of 0–2 M at temperatures near the melting temperature by DSF. The unfolding fraction in the presence of urea was converted to an equilibrium constant and ΔG _U at each temperature. ΔG _U (H₂O) was determined using the linear extrapolation method. The T _m of 3α‐HSD/CR was 51.6°C and 39.6°C in 0 and 2 M urea, respectively. The thermodynamic parameters obtained at temperatures lower than 40°C and higher than 51°C were less reliable because less data was available for the transition region of thermal unfolding. The values of ΔG _U (H₂O), m, and [urea]_0.5 from temperature 41°C to 50°C are shown in Table 2. As expected, the values of ΔG _U (H₂O) and [urea]_0.5 decreased as the temperature increased. These consistent results from different approaches for determining ΔG _U (T) at pH 7.6 are presented in Figure 6b. This study provides an alternate determination of ΔG _U (H₂O) via urea‐induced denaturation at temperatures near the transition region of thermal unfolding, as well as a reduction in uncertainty in obtaining ΔG _U (H₂O) from the long extrapolation of the transition region of protein denaturation induced by urea. Taken together, the unfolding of 3α‐HSD/CR was induced by heat and denaturants, such as urea, GdmCl, and protons, and its progress was monitored by the change in fluorescence intensity detected by fluorometry or RT‐PCR. These probes yielded nearly identical G _U (T) values.

The protein stability curve showed maximum stability with thermal unfolding at high temperatures and predicted cold unfolding at low temperatures, causing the ΔG _U to be zero at T _m and T _c , respectively. The values of T _m and the predicted T _c of 3α‐HSD/CR at pH 7.6 are 51.6°C and 2.2°C and change to 38.3°C and 13.5°C at pH 4.5, respectively. However, we did not observe cold denaturation of 3α‐HSD/CR from 25°C to 6°C, pH 4.5 by measuring intrinsic tryptophan fluorescence. The maximum intrinsic fluorescence of 3α‐HSD/CR remained at 330 nm (λ_max) at 6°C, pH 4.5, similar to that of the native enzyme measured at 25°C, pH 7.6. Maximum stability in the protein stability curve occurred at temperature T _s , where ΔS _U was equal to zero. T _s was calculated using the equation $T_s=T_m/\exp \left[∆H_{m,\text{calc}}/\left(T_m∆C_p\right)\right]$, where T _m in Kevin (Becktel & Schellman, 1987). The T _s is 26.4°C at pH 7.6, where the maximum stability of 3α‐HSD/CR is only contributed by ΔH _U of 30.79 kJ mol⁻¹ and decreases to 25.7°C with ΔH _U of 7.74 kJ mol⁻¹ at pH 4.5 (the dotted line in Figure 6b). Meanwhile, when ΔH _U is equal to zero kJ mol⁻¹ at temperature T _h , the protein is only stabilized by the entropic contribution, that is, ΔG _U  = −T _h ΔS. The value of T _h can be calculated from the equation of $T_h=T_m-\left(∆H_m/∆C_p\right)$, giving 25.5°C with ΔG _U of 30.75 and 7.74 kJ mol⁻¹ at pH 7.6 and pH 4.5, respectively. The difference of 23.01 kJ mol⁻¹ in denaturation was mainly due to the loss of ΔS as the pH increases from 4.5 to 7.6.

We analyzed the protein stability curve to characterize the stability of 3α‐HSD/CR, giving a ΔG _U of 30.67 kJ mol⁻¹ for 3α‐HSD/CR at pH 7.6, 25°C. ΔG _U decreases from 30.67 to 7.70 kJ mol⁻¹ as the pH decreases from 7.6 to 4.5 at 25°C (Figure 6b). The contribution of enthalpy and entropy to protein stability were calculated, yielding ΔH _U and TΔS _U of −16.11 and −46.78 kJ mol⁻¹ at pH 7.6, 25°C, respectively, indicating that the change of entropy mainly contributes to the protein folding in the native state. Additionally, the negative value of the change in entropy with protein unfolding suggests that the hydrophobic effect causes the order of H₂O to surround the exposed nonpolar residues and leads to a decrease in entropy. Hydrophobic interactions favor the burial of hydrophobic residues in the core of the protein. As the temperature increased, the clathrate structures became weaker than bulk water, the randomness of the unfolding of protein increased, and the value of S_U became positive, favoring protein unfolding. Therefore, denaturation entropy was favorable for the thermal denaturation of 3α‐HSD/CR.

The protein stability affected by pH can result from the differential ionization of amino acid residues in native and denatured states (Graziano et al., 1999; Pace et al., ²⁰⁰⁹; Tollinger et al., ²⁰⁰³). A buried ionizable amino acid can be titrated differently by acids in folded and unfolded conformations of the protein. The pK_a values of the ionizable groups in proteins are affected by interactions with the surrounding environment, such as nonpolar, charge, and H‐bonding interactions, causing a shift in the pK_a values of the residues in the folded protein from its standard value. As pH decreased, the buried ionizable group took up protons when exposed to the denatured state and decreased ΔG _U . The linked equilibrium between protein denaturation and protonation for 3α‐HSD/CR is shown in Figure 6c, and the relationship between conformational stability and differential pK_a of amino acids in the native and denatured states is represented by Equation (5). We considered that the differential ionization of amino acid residues in the native and denatured states could result in protein destabilization as the pH decreased. Residues that bind protons more tightly in the unfolded conformation than in the native state stabilize the protein as proton concentration decreases (Tollinger et al., 2003). Therefore, we determined the number of residues with differential ionization constants in the folded and unfolded states that contribute to the pH‐dependent stability of 3α‐HSD/CR. The study on pH‐dependent protein stability shows that ΔG _U decreases from 30.67 to 7.70 kJ mol⁻¹ as the pH decreases from 7.6 to 4.5 at 25°C. The number of proton uptakes or releases (ΔQ) at each pH was determined by applying Equation (4) to the data in Table 1, giving a maximum of two proton uptakes during protein unfolding at pH 4.5–7.6. Therefore, the decrease in the thermodynamic stability of dimeric 3α‐HSD/CR with decreasing pH suggests that the uptake of protons from a residue at each monomer is coupled to the conformational unfolding transition.

We assumed that the binding sites for the residue at each monomer were identical and non‐interacting, with different association constants in the native and denatured states (Graziano et al., 1999). The pK_a obtained from pH‐dependent protein stability are 4.0 and 6.5 for residues in native and denatured states, respectively. The denatured state of a protein is treated as a state in which all ionizable groups titrate independently of their standard pK_a values. A decrease in the pK_a shift of an ionizable residue in a native protein is usually caused by electrostatic interactions between neighboring positive charges or by desolvation effects in a hydrophobic environment (Oliveberg et al., 1995). The pK_a of His in solution is 6.5 (Grimsley et al., 2009). His238 is located at the dimeric interface. We deduced that the His238 residue of 3α‐HSD/CR with apparent pK_a of 4.0 and 6.5 in the native and denatured states, respectively, may play a role in pH‐dependent stability. It is assumed that ΔH _U does not change as pH decreases (−16.11 kJ mol⁻¹ at 25°C). Thus, the increases in ΔG _U from 7.70 to 30.67 as the pH increases from 4.5 to 7.6 are due to the decrease in the change of entropy (TΔS _U ) during unfolding from −23.81 to −46.78 kJ mol⁻¹, respectively. The deprotonation of the His residue upon unfolding could increase the order of water surrounding the His residue, resulting in a decrease in entropy gain, thus increasing the ΔG _U as the pH increases_. We are currently studying the pH dependence of protein stability in His‐residue mutants.

4. CONCLUSIONS

The pH‐dependent protein stability curve of 3α‐HSD/CR was determined by fitting the values of ΔH _m , T _m , and ΔC _p using the Gibbs–Helmholtz equation at various temperatures, in combination with the ΔG _U obtained from denaturant‐induced denaturation by the linear extrapolation method at various temperatures. The thermodynamic parameters ΔH _m and T _m were determined by the thermal unfolding of 3α‐HSD/CR using DSF, whereas the value of ΔC _p was determined by a linear relationship between ΔH _m and T _m perturbed by either pH or urea. To obtain ΔG _U at temperatures near the melting temperature, we developed an alternative method to determine ΔG _U (H₂O) from the pretransition and transition regions of protein unfolding induced by urea at various temperatures using DSF, which gave values similar to those obtained from the protein stability curves.

The maximum stability of 3α‐HSD/CR is mainly caused by the change of entropy (−TΔS _U ) with 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 from the pH‐dependent protein stability curve. The analysis of ΔG_D at varied pH with the linked equilibrium between protein denaturation and protonation indicated that two proton uptakes from residues with pK_a of 4.0 and 6.5, in native and denatured states, respectively, are involved in protein unfolding as the pH decreases from 7.6 to 4.5 at 25°C. ΔG _U comprises ΔH _U and −TΔS _U , which depend on ΔC _p between the denatured and native states of the protein. We determined ΔC _p of 30.25 kJ mol⁻¹ K⁻¹ for the denaturation of 3α‐HSD/CR, suggesting that hydrophobic interactions play an important role in the folding of 3α‐HSD/CR. As 3α‐HSD/CR unfolds, the exposed nonpolar residues are surrounded by water, forming clathrate structures. This ordered water structure requires more energy to break down, resulting in an increase in heat capacity and a decrease in the change in entropy upon protein unfolding. As the temperature increased, the randomness of the protein unfolding increased. Therefore, the entropy of denaturation was favorable for the thermal denaturation of 3α‐HSD/CR. This study established an alternative methodology to determine ΔG _U (H₂O) from the pretransition and transition regions of protein unfolding induced by urea at various temperatures near T _m through DSF. The energetics of the stability of 3α‐HSD/CR using DSF in combination with conventional denaturant‐induced denaturation were characterized in detail and provided insights into the structure–function relationship and enzyme activity of HSD enzymes.

5. MATERIALS AND METHODS

5.1. Reagents

Comamonas testosteroni 3α‐HSD/CR was overexpressed in BL21(DE3) cells and purified as previously described (Hwang et al., 2005). The purified protein was confirmed to be homogeneous by SDS‐PAGE. Protein concentrations were determined spectrophotometrically from the UV absorbance at 280 nm using a dimeric form with ε₂₈₀ = 22,920 M⁻¹ cm⁻¹, which was calculated using ProtParam (Walker, 2005). The buffers used were from Sigma (Mes hydrate, Hepes, Ches, Taps, Caps) and BIO BASIC Inc. (acetate). The pH of the solutions was measured at room temperature after a double buffer adjustment on a Radiometer Analytical PHC4000‐8 Combination Calomel pH electrode. SYPRO Orange 5000× was purchased from Thermo Fisher Scientific.

5.2. Thermal unfolding of 3α‐HSD/CR by DSF

pH‐dependent thermal unfolding of 3α‐HSD/CR was performed using DSF. Typically, a solution of 2 μM 3α‐HSD/CR with 5× SYPRO Orange in 100 mM buffer at various pH values was added to a 96‐well PCR plate and sealed with an optical adhesive film to prevent evaporation. The buffer used for the pH‐dependent study was acetate, pH 4.5–5.5; Mes, pH 5.5–7.0; Hepes, pH 7.0–8.5; Taps, pH 8.5–9.0; and Ches, pH 9.0–10.0. The sample solution in the plate was shaken using a plate reader at 270 cpm for 1 min to mix homogeneously and centrifuged at 1500g for 5 min to remove any air bubbles present in the solution before measuring the fluorescence. Thermal unfolding in the presence of 0–2 M urea was performed in 100 mM Hepes, pH 7.6. A StepOne Plus system RT‐PCR (Applied Biosystems) was used to collect DSF data using the ROX channel for fluorescence excitation and emission. The heating rate used was 0.5°C min⁻¹. The fluorescence intensity was recorded from 25°C to 85°C. The data files obtained from the RT‐PCR runs were imported into a Microsoft EXCEL spreadsheet and further analyzed with SigmaPlot 11.0, using the appropriate equations. The baselines of the native and denatured states were obtained by selecting from the linear regions of the low and high temperatures, respectively, and were included in the fitting procedure. The fluorescence intensity data were fitted to Equation (1) to determine the T _m and ΔH _m values of 3α‐HSD/CR.

5.3. Denaturant‐induced denaturation of 3α‐HSD/CR

The Gibbs free energy of denaturation of 3α‐HSD/CR was determined in the presence of denaturants at various temperatures. 2 μM 3α‐HSD/CR was incubated overnight with various concentrations of urea or GdmCl in 100 mM Hepes at pH 7.6. Stock solutions of urea and GdmCl were freshly prepared, and the concentration was determined as described by Pace (Pace, 1986). Fluorescence emission was monitored using a Perkin‐Elmer LS55 luminescence spectrometer with excitation at 295 nm and emission at 300–450 nm. The intrinsic fluorescence intensity at 317 nm was plotted as a function of denaturant concentration and analyzed using a two‐state equilibrium unfolding model to obtain ΔG _D (H₂O), m, and [D]_0.5.

5.4. Data analysis

The temperature dependence of the stability of 3α‐HSD/CR at various pH values was investigated using DSF. The two‐state model of the native (N) and denatured states (D) associated with the equilibrium constant (K _U ) and the free energy of denaturation (ΔG _U ) are shown in Figure 6a. Protein denaturation was induced by temperature or denaturants following changes in fluorescence intensity. Data from the observed fluorescence intensity (F _obs) as a function of temperature (T) were fitted to Equation (1) to obtain the thermodynamic parameters T _m and ΔH _m . T _m is the temperature at which 50% of the protein is in the denatured state and ΔH _m is the enthalpy of denaturation at T _m . R is the gas constant, and T is the temperature in Kelvin. F _N (T) and F _U (T) are assumed to be linear functions of temperature, that is, $F_N\left(T\right)=F_{\mathrm{N}0}+\left(N_s\times T\right)$ and $F_U\left(T\right)=F_{\mathrm{U}0}+\left(U_s\times T\right)$, respectively. N _s and U _s are the changes in the fluorescence intensities of N and U with respect to temperature, respectively, and F _N0 and F _U0 are the intercepts of the fluorescence intensities of N and U, respectively. ΔC _p is the heat capacity change associated with protein unfolding, and is determined from the linear relationship between ΔH _m and T _m , as shown in Equation (2). To analyze the change in the free energy in the two‐state protein denaturation process at various temperatures, ΔG _U (T) was calculated as a function of temperature using the Gibbs–Helmholtz equation (Equation (3)). Thus, the protein stability curve as a function of temperature was plotted using Equation (3) with the experimentally determined thermodynamic parameters ΔH _m , T _m , and ΔC _p .

The pK_a of the residues and the number of proton uptakes during unfolding were determined by analyzing pH‐dependent protein stability. The electrostatic contributions to protein stability can be perturbed by varying the pH, owing to the differential pK_a values of the ionizable groups between the folded and unfolded states. The difference in the number of bound protons between the native and denatured states can be related to the pH dependence of protein stability shown in Equation (4), where ΔQ is the difference in the number of bound protons between the native and denatured states. The link between binding with two protons and conformational unfolding transitions for the two‐state model and the equilibrium constants are shown in Figure 6c, where N and HNH are the deprotonated and protonated native states, respectively, and U and HUH are the deprotonated and protonated denatured states, respectively. The binding sites for the residues were assumed to be identical and non‐interacting with different association constants in the native and denatured states. Thus, K _a‐NH and K _a‐UH are the ionization constants of the residues involved in pH‐dependent denaturation in the native and denatured states, respectively. where K _U and K _U‐NH are the equilibrium constants for the native and denatured states with deprotonation and protonation, respectively. K _U (pH) is the pH‐dependent equilibrium constant. The Gibbs free energy ΔG _U (pH) for the residues involved in pH‐dependent protein denaturation with binding (or releasing) of the two protons is shown in Equation (5). ΔG _U (pH) can be divided into ΔG _U‐NH and ΔG _U‐pH. ΔG _U‐NH represents a pH‐independent term that includes non‐electrostatic and electrostatic contributions at pH where an ionizable residue is protonated in both states. ΔG _U‐pH is related to protonation‐deprotonation equilibria involving individual ionizable groups.

The stability of 3α‐HSD/CR in the presence of urea was determined using DSF. We converted the observed fluorescence intensity (F _obs) in the transition region of the unfolding curve in the presence of urea to ΔG _U (D) using Equation (6) at a fixed temperature, where F_N and F_U were obtained by extrapolating the pre‐ and post‐transition fluorescence intensities of N and U into the transition region, respectively. Data from ΔG _U (D) were fitted to Equation (7) to determine the Gibbs free energy of denaturation in the absence of denaturant (D), ΔG _U (H₂O), and m values. m is the dependence of the Gibbs free energy of denaturation on the denaturant concentration. Protein denaturation induced by denaturants at various temperatures was also performed using a spectrofluorometer. The observed fluorescence intensity in the presence of denaturant at various temperatures was fitted to Equation (8) to obtain the ΔG _U (H₂O) and m values.

$$F_{\mathrm{obs}}=\frac{F_N\left(T\right)+\exp -\left\{\frac{∆H_m\left(1-\frac{T}{T_m}\right)+∆C_p\left[\left(T-T_m\right)-T\ln \left(\frac{T}{T_m}\right)\right]}{\mathit{RT}}\right\}\times F_D\left(T\right)}{1+\exp -\left\{\frac{∆H_m\left(1-\frac{T}{T_m}\right)+∆C_p\left[\left(T-T_m\right)-T\ln \left(\frac{T}{T_m}\right)\right]}{\mathit{RT}}\right\}}$$ (1)

$$∆C_p=\frac{\delta ∆H}{\mathit{\delta T}}$$ (2)

$$∆G_U\left(T\right)=∆H_m\left(1-\frac{T}{T_m}\right)+∆C_p\left[\left(T-T_m\right)-T\ln \left(\frac{T}{T_m}\right)\right]$$ (3)

$$d∆G_U/\mathit{dpH}=2.3\mathit{RT}∆Q$$ (4)

$$∆G_U\left(\mathit{pH}\right)=∆G_{U-\mathit{NH}}+∆G_{U-\mathit{pH}}$$

$$=-\mathit{RT}\ln K_{U-\mathit{NH}}-\mathit{RT}\ln {\left\{\frac{\left[H+K_{a-\mathit{UH}}\right]}{\left[H+K_{a-\mathit{NH}}\right]}\right\}}^2$$ (5)

$$∆G_U\left(\mathrm{D}\right)=-\text{RTln}\left[\left(F_{\mathrm{obs}}-F_N\right)/\left(F_U-F_{\mathrm{obs}}\right)\right]$$ (6)

$$∆G_U\left(D\right)=∆G_U\left(H_{2}O\right)-m\left[D\right]$$ (7)

$$F_{\mathrm{obs}}=\frac{\left\{F_N+F_U\exp \left(-\left(\frac{∆G_D^{H2O}-m\left[D\right]}{\mathit{RT}}\right)\right)\right\}}{\left\{1+\exp \left(-\left(\frac{∆G_D^{H2O}-m\left[D\right]}{\mathit{RT}}\right)\right)\right\}}$$ (8)

AUTHOR CONTRIBUTIONS

Yun‐Hao Chou: Conceptualization; investigation (lead); data curation; formal analysis (lead); writing—original draft (supporting). Yan‐Liang Chen: Investigation. Chia‐Lin Hsieh: Investigation. Tzu‐Pin Wang: Conceptualization (supporting). Wen‐Jeng Wu: Conceptualization (supporting). Chi‐Ching Hwang: Conceptualization (lead); methodology; supervision; validation, writing—original draft (lead); formal analysis (lead); writing—original draft (lead); writing—review and editing (equal).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

FIGURE S1. The fluorescence intensity of thermal unfolding of 3α‐HSD/CR by DSF at pH 2.0–4.5. The fluorescence intensity of thermal unfolding of 3α‐HSD/CR significantly decreased when the pH decreased from 4.0 to 2.0. The higher fluorescence intensity at lower temperatures at pH 3.5 and 4.0 is mainly due to the acid‐induced unfolding of 3α‐HSD/CR, while the lower fluorescence intensity at pH 2.0–3.0 is due to the quenching of the fluorescence of SYPRO Orange. 2 μM 3α‐HSD/CR with 5× SYPRO orange in 100 mM buffer at the indicated pH was heated at 0.5°C min⁻¹ from 25 to 85°C using DSF. The buffers used in the study were glycine, pH 2.0–3.5; formate, pH 3.5–4.0; acetate, pH 4.0–4.5.

FIGURE S2. Thermal unfolding of 3α‐HSD/CR at various concentrations and scan rates using DSF. (a) The thermal unfolding of 1–12 μM 3α‐HSD/CR by DSF shows a sigmoidal curve at pH 7.1. The solid lines represent the fit of the data to Equation (1) to obtain T _m and ΔH _m . (b, c) T _m and ΔH _m values for 3α‐HSD/CR at various concentrations, respectively, and showed independent on the concentration of 3α‐HSD/CR. Data are mean ± SD of at least three independent experiments. (d, e) The T _m and ΔH _m of 3α‐HSD/CR at pH 4.5–7.6 were determined by DSF and showed similar values at scan rates of 0.5–2.0°C min⁻¹.

FIGURE S3. Intrinsic fluorescence spectra of 3α‐HSD/CR in the presence of urea and GdmCl at various temperatures. (a) Fluorescence emission spectra of 3α‐HSD/CR denaturation induced by urea at 15°C, 22°C, 25°C, and 30°C, and (b) induced by GdmCl at 22°C, pH 7.6. 3α‐HSD/CR was excited at 295 nm and the emission was measured from 300 to 450 nm using a spectrophotometer. λ_max shifted from 330 to 355 nm as the concentration of urea increased from 0 to 6 M, and GdmCl from 0 to 3 M.

TABLE S1. T _m and ΔH _m values of 3α‐HSD/CR using DSF at pH 7.8–9.7^a

TABLE S2. T _m and ΔH _m values of 3α‐HSD/CR in the presence of 0–2 M urea at pH 7.6 by DSF^a

Chou Y‐H, Hsieh C‐L, Chen Y‐L, Wang T‐P, Wu W‐J, Hwang C‐C. Characterization of the pH‐dependent protein stability of 3α‐hydroxysteroid dehydrogenase/carbonyl reductase by differential scanning fluorimetry. Protein Science. 2023;32(8):e4710. 10.1002/pro.4710