Human aldose reductase unfolds through an intermediate.

Materials

All chemicals were reagent grade and purchased from Sigma-Aldrich.

Protein purification

The hAR cDNA cloned into expression vector pET-15b (Novagen) was a kind gift from Dr. Alberto Podjarny (Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, France). The plasmid, coding for a hexahistidine-tagged protein, was expressed into E. coli strain BL21 (DE3) (Novagen). The cells with recombinant plasmid were grown with 100 μM ampicillin at 37°C to an OD₆₀₀ nm value of 0.7 and protein expression was induced by adding 1 mM IPTG. Cells were grown for further 3 hours at 37°C. All further operations were carried out at 4°C unless otherwise stated. Cells were centrifuged, re-suspended and lysed by sonication. A Ni-NTA affinity column (GE Healthcare) was used for protein purification. The material used for stationary phase for the column was Ni-Sepharose and the flow rate of column was adjusted to 0.5 ml min^-1. Imidazole and other salts were removed by repeated dialysis in 50 mM potassium phosphate, pH-7 buffer containing 50 mM NaCl. Protein concentration was estimated using the molar extinction coefficient and absorbance reading at 280 nm. The histidine tag from recombinant protein was removed by thrombin (4 units of thrombin per mg of recombinant protein at room temperature for 3 hours). Cleaved protein was passed through the Ni-NTA column, and purified protein without tag was collected as flow-through. Enzyme activity was checked as per standard assay¹⁰. Homogeneity and molecular weight of hAR with and without histidine tags was analyzed under denaturing conditions on 15% SDS-PAGE. Purified hAR was stored at -20°C for further studies.

Thermal unfolding

Thermal unfolding was carried out at a final concentration of 2.8 µM protein in 50 mM potassium phosphate buffer, pH 7.0 containing 50 mM NaCl and 0.1mM TCEP. Transition between 20–70 °C was followed using a far-UV circular dichroism (CD) signal at 222 nm by using 0.1 cm path length cuvette at sampling rate of 1.0 °C min^-1 in a Jasco J-810 spectropolarimeter. Buffer blank was duly subtracted before reporting the change in ellipticity (millidegree) at 222 nm.

Chemical induced unfolding

Samples with 1.4 μM protein concentration were prepared in phosphate buffer (described earlier) containing different concentrations of GuHCl/urea. Samples were incubated for 12 hours to reach equilibrium at 25°C after which no change in signal occurred either in fluorescence or CD spectra. Trp fluorescence (excitation at 295 nm and emission recorded between 300 nm to 400 nm) and ANS fluorescence (excitation at 370 nm and emission recorded between 400 nm to 600 nm) measurements were performed using a Hitachi F-7000 fluorescence spectrophotometer for GuHCl samples and Jasco J-815 spectropolarimeter for urea samples. Far-UV CD measurements were performed using Jasco J-810 spectropolarimeter for GuHCl samples and Jasco J-815 spectropolarimeter for urea samples. Quartz cuvette of 1 cm and 0.5 cm path length were used for fluorescence and CD measurements respectively. All measurements were done at 25 °C. Spectra were reported as ellipticity (millidegree) after baseline correction.

Heat capacity change (ΔC_p) calculations

ΔC_p value for unfolding of hAR was calculated from change in accessible surface area (ΔASA) according to Equation 1¹¹

$\Delta C_p=-251+0.19\times [\Delta ASA](\mathrm{Equation\; 1})$

ProtSA web server was used to calculate change in accessible surface area from native to unfolded conformation of hAR¹².

Data analysis

GraphPad Prism version 7.04 for Windows (GraphPad Software, La Jolla, California) was used for analysis of thermal and chemical induced unfolding data on the basis of two and three state model respectively as described in following sections.

Thermal unfolding

Data was fitted by least square analysis to Equation 2¹³.

$Y=\frac{(A_n+b_N\times T)+(A_u+b_U\times T)\times exp\left(\left(\frac{\Delta H_g}{R}\right)\times \left(\frac{1}{T_g}-\frac{1}{T}\right)\right)}{1+\exp \left(\left(\frac{\Delta H_g}{R}\right)\times \left(\frac{1}{T_g}-\frac{1}{T}\right)\right)}(\mathrm{Equation\; 2})$

Where A_n and A_u are native and unfolded state baseline intercepts respectively and b_N and b_U are native and unfolded baseline slopes respectively. ΔH_m is enthalpy change at melting temperature (T_g). T is absolute temperature and R is the gas constant.

Calculation of ∆G values for transition region

Signal for native (Y_N = A_n + b_N × T) and unfolded baseline (Y_U = A_u + b_U × T) for every point in transition region was calculated from Equation 2. If Y is signal for a particular point in transition region, then fraction of unfolded protein (F_u) at this point is given by Equation 3.

$F_u=\frac{Y_n-Y}{Y_n-Y_u}(\mathrm{Equation\; 3})$

The equilibrium constant (k) can be calculated from relative population of species using Equation 4.

$k=\frac{F_u}{1-F_u}(\mathrm{Equation\; 4})$

ΔG can be calculated as a function of temperature using Equation 5.

$\Delta G=-RTlnk(\mathrm{Equation\; 5})$

Thermal stability curve

The thermal stability curve of hAR was constructed on the basis of Equation 6–8¹⁴.

$\Delta H_T=\Delta H_g+\Delta C_p(T-T_g)(\mathrm{Equation\; 6})$

$\Delta S_T=\frac{\Delta H_g}{T_g}+\Delta C_p\times LN\left(\frac{T}{T_g}\right)(\mathrm{Equation\; 7})$

$\Delta G_s=\Delta H_g\times \left(1-\frac{T}{T_g}\right)+\Delta C_p\times \left(T-T_g-\left(T\times LN\left(\frac{T}{T_g}\right)\right)\right)(\mathrm{Equation\; 8})$

Where ΔH_T and ΔS_T are enthalpy and entropy change respectively at temperature T with reference to T_g. T_h, T_s and T_g are the temperatures at which ∆H, ∆S, and ∆G are zero respectively. ∆G_s is the stabilization free energy of the native state relative to the unfolded state.

Chemical induced unfolding

Data was fitted by least square analysis to Equation 9¹³.

$Y=\frac{(A_n+b_N\times [D])+(A_i+b_I\times [D])\times \exp \left(-\left(\frac{\Delta G_{(N-I)}}{R*T}-\frac{m_{{(N-I)}^{\times [D]}}}{R*T}\right)\right)+(A_u+b_U\times [D])\times \exp \left(-\left(\frac{\Delta G_{(N-I)}}{R\times T}-\frac{m_{{(N-I)}^{\ast [D]}}}{R\times T}\right)\right)\times \exp \left(-\left(\frac{\Delta G_{(I-U)}}{R\times T}-\frac{m_{{(I-U)}^{\times [D]}}}{R\times T}\right)\right)}{1+\exp \left(-\frac{\Delta G_{(N-I)}}{R\times T}-\frac{m_{{(N-I)}^{\times [D]}}}{R\times T}\right)-\exp \left(-\left(\frac{\Delta G_{(N-I)}}{R\times T}-\frac{m_{{(N-I)}^{\times [D]}}}{R\times T}\right)\right)\times \exp \left(-\left(\frac{\Delta G_{(I-U)}}{R\times T}-\frac{m_{{(I-U)}^{\times [D]}}}{R\times T}\right)\right)}(\mathrm{Equation\; 9})$

Where A_n, A_u, and A_i are the native, unfolded and intermediate baseline intercepts, respectively, and b_N, b_U and b_I are the native, unfolded and intermediate baseline slopes, respectively. [D] is denaturant concentration in molar. m_(N-I) and m_(I-U) are denaturant gradient for native to intermediate and intermediate to unfolded state respectively. ∆G_(N-I) and ∆G_(I-U) are stabilization free energy of intermediate state relative to native and unfolded state respectively.

Thermal unfolding monitored by far-UV CD

Change in ellipticity at 222 nm fitted well on the basis of a two-state model (Figure 1A). This analysis gave values for ΔH_g and T_g which along with ΔC_p value calculated from Equation 1 were used for non-linear regression of transition region (±5 kJ mol^-1) to Equation 8 (Figure 1C). Values of ΔH and ΔS were calculated over extended range of temperature by using Equation 7 and Equation 8, respectively (Figure 1D). Thermal stability curve is extrapolation of transition region assuming constant ΔC_p during unfolding transition (Figure 1E). The relationship between T_s, T_h and ∆G (T_s – T_h = ∆G_s/∆C_p) is presented in Figure 1F. Thermodynamic parameters obtained from analysis of thermal unfolding data are listed in Table 1. All raw data are available as Underlying data¹⁵.

Figure 1. Thermal unfolding studies of human aldose reductase (hAR) monitored by far-UV circular dichroism.

(A) Change in ellipticity at 222 nm plotted as a function of temperature. (B) Fraction of protein folded (green dots) and unfolded (red dots) plotted against temperature. (C) Portion of transition curve used in van't Hoff analysis. (D) Plots of ΔH and ΔS as function of temperature. (E) thermal stability curve of hAR. (F) Triangular relationship among T_h, T_s and ∆G_s. Explanation for T_g, T_g’, T_h and T_s is given in text. Dashed lines are extrapolations. Solid line represents fit to unfolding transition, filled symbols represent data points from unfolding experiments and red symbols represent outliers.

Chemical induced unfolding monitored by fluorescence

There are six Trp residues in hAR, out of which four are part of the hydrophobic active site pocket in the core of the β-barrel and two are buried in alpha helices surrounding the barrel. Their fluorescence provided global signal of change in tertiary structure. ANS has been extensively used as a probe for non-native, partially unfolded conformations of protein. The binding of ANS to hydrophobic regions results in a significant enhancement of ANS fluorescence and a pronounced blue-shift of the λ_max¹⁶.

Fluorescence emission profiles of hAR equilibrated with different concentrations of denaturants are presented in Figure 2 (Figure 2A and Figure 3A for urea and GuHCl, respectively). A plot of λ_max against denaturant concentration indicated cooperative transition from native to unfolded state (Figure 2B and Figure 3B for urea and GuHCl, respectively). In case of ANS fluorescence, significant blue-shift of around 20 nm and 10 nm from the native to intermediate state was observed for urea and GuHCl, respectively (Figure 2B2 and Figure 3B2 for urea and GuHCl, respectively).

Figure 2. Urea induced unfolding studies of human aldose reductase (hAR)monitored by fluorescence.

(A1) Trp fluorescence scans and (A2) ANS fluorescence scans for all the samples. (B1) λ_max (Trp fluorescene) and (B2) λ_max (ANS fluorescence) against [urea]. (C1) I_max (Trp fluorescenc) and (C2) I_max (ANS fluorescence) against [urea]. (D1) I_295/314 (Trp fluorescence) and (D2) I_370/480 (ANS fluorescence) against [urea]. (E1) Trp fluorescence and (E2) ANS fluorescence of samples in native (green), intermediate (cyan) and unfolded state (red). Solid lines represent fit to the unfolding transitions, filled symbols represent data points from unfolding experiments, red symbols represent outliers.

Plot of I_max against denaturant concentration indicated presence of an intermediate during unfolding transition (Figure 2C and Figure 3C for urea and GuHCl, respectively). I_max in case of ANS fluorescence fits satisfactorily on the basis of three-state model (Figure 2C2 and Figure 3C2 for urea and GuHCl, respectively). For both urea and GuHCl induced unfolding, Trp fluorescence emission intensity at 314 nm fits satisfactorily to three-state model. In case of ANS fluorescence, both I_max and fluorescence emission intensity at 480 nm fit equally well on the basis of three-state model. Thus, Trp fluorescence emission intensity at 314 nm and ANS fluorescence emission intensity at 480 nm were analyzed on the basis of three-state model to evaluate thermodynamic stability of hAR (Figure 2D and Figure 3D for urea and GuHCl, respectively). The thermodynamic parameters obtained from fitting are listed in Table 1. Trp and ANS fluorescence clearly demonstrate presence of an intermediate state populated at 3.5-4.0 M and 0.7-2 M urea and GuHCl concentration respectively, apart from the native and unfolded states (Figure 2E and Figure 3E for urea and GuHCl, respectively).

Figure 3. GuHCl-induced unfolding studies of human aldose reductase (hAR)monitored by fluorescence.

(A1) Trp fluorescence scans and (A2) ANS fluorescence scans. (B1) λ_max (Trp fluorescence) and (B2) λ_max (ANS fluorescence) against [GuHCl]. (C1) I_max (Trp fluorescence) and (C2) I_max (ANS fluorescence) against [GuHCl]. (D1) I_295/314 (Trp fluorescence) and (D2) I_370/480 (ANS fluorescence) against [GuHCl]. (E1) Trp fluorescence and (E2) ANS fluorescence of samples in native (green), intermediate (cyan) and unfolded state (red). Solid lines represent fit to the unfolding transitions, filled symbols represent data points from unfolding experiments, red symbols represent outliers.

Chemical induced unfolding monitored by far-UV CD

Unfolding profiles of hAR equilibrated at different denaturants concentrations in far-UV CD are presented in Figure 4A1 and 4A2 for GuHCl and urea, respectively. Thermodynamic stability of hAR was determined on the basis of three state model by plotting change in ellipticity at 219/222 nm as a function of denaturant concentration (Figure 4B1 and 4B2 for GuHCl and urea, respectively). The transition determined by far-UV CD detected intermediate state at similar concentrations of denaturant as interrogated by fluorescence spectroscopy. All three states can be clearly distinguished from Far-UV CD profiles (Figure 4C1 and 4C2 for GuHCl and urea, respectively). Thermodynamic parameters derived from far-UV CD data are listed in Table 1.

Figure 4. GuHCl/urea induced unfolding studies of human aldose reductase (hAR)monitored by far-UV circular dichroism (CD).

(A1) far-UV CD scans recorded for GuHCl and (A2) far-UV CD scans recorded for urea for all the samples. (B1) change in ellipticity at 219 nm against [GuHCl] and (B2) change in ellipticity at 221.8 nm against [urea]. (C) CD spectra of samples in native (green), intermediate (cyan) and unfolded state (red) for (C1) GuHCl and (C2) urea, respectively. Solid line represents fit to unfolding transitions, filled symbols represent data points from unfolding experiments and red symbols represent outliers in data fitting.

Underlying data

Figshare: data_f1000_hAR_unfolding.zip. https://doi.org/10.6084/m9.figshare.8001998.v1¹⁵.

This project contains raw data for chemically and thermally induced unfolding studies on human aldose reductase.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).