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 a further 3 hours at 37°C. All further operations were carried out at 4°C unless otherwise stated. Cells were centrifuged, resuspended, and lysed by sonication. A Ni-NTA affinity column (GE Healthcare) was used for protein purification. The material used for the stationary phase for the column was Ni-Sepharose, and the flow rate of the 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). The 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 assay¹³. We analyzed the homogeneity and molecular weight of hAR with and without histidine tags under denaturing conditions on 15% SDS-PAGE. Purified hAR was stored at -20°C for further studies.

Thermal denaturation

Thermal denaturation 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. The transition between 20–70 °C was followed using a far-UV circular dichroism (CD) signal at 222 nm by using a 0.1 cm path-length cuvette at a sampling rate of 1.0 °C min^-1 in a Jasco J-810 spectropolarimeter. After subtracting buffer blank, we have reported the change in ellipticity (millidegree) at 222 nm.

Chemical induced unfolding

To follow chemical induced unfolding, we prepared samples with 1.4 μM protein concentration 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. We have reported spectra as ellipticity (millidegree) after baseline correction.

Heat capacity change (ΔC_p) calculations

We have calculated the value of ΔC_p for the unfolding of hAR from the change in accessible surface area (ΔASA) according to Equation 1¹⁴

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

ProtSA webserver was used to calculate the change in accessible surface area from native to the unfolded conformation of hAR¹⁵.

Data analysis

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

Thermal denaturation

The Least-square analysis was used to fit the data to Equation 2¹⁶.

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

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 ΔG values for the 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 the 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) was calculated from the relative population of species using Equation 4.

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

ΔG was 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 based on Equation 6–8¹⁷.

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

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

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

Where ΔH_T and ΔS_T are enthalpy change and entropy change, respectively, at temperature T with reference to T_g. T_h, T_s and T_g are the temperatures at which ΔH, ΔS, and ΔG are zero, respectively. ΔG_s is the stabilization free energy of the native state relative to the unfolded state.

Chemical induced unfolding

The least-square analysis was used to fit the data to Equation 9¹⁶.

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

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 a molar unit. m_(N-I) and m_(I-U) are denaturant gradient for native to intermediate and intermediate to unfolded state, respectively. ΔG_(N-I) and ΔG_(I-U) are stabilization free energy of intermediate state relative to the native and unfolded state, respectively.

Thermal denaturation monitored by far-UV CD

Change in ellipticity at 222 nm fitted well based on a two-state model (Figure 1A). This analysis gave values for ΔH_g and T_g, which along with the ΔC_p value calculated from Equation 1 were used for non-linear regression of the transition region (±5 kJ mol^-1) to Equation 8 (Figure 1C). Values of ΔH and ΔS were calculated over an extended range of temperatures by using Equation 7 and Equation 8, respectively (Figure 1D). The thermal stability curve is an extrapolation of transition region, assuming constant ΔC_p during the unfolding transition (Figure 1E). The relationship between T_s, T_h and ΔG (T_s – T_h = ΔG_s/ΔC_p) is presented in Figure 1F. Thermodynamic parameters obtained from the analysis of thermal denaturation data are listed in Table 1. All raw data are available as Underlying data¹⁸.

Figure 1. Thermal denaturation 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) The fraction of protein folded (black dots) and unfolded (cyan dots) plotted against temperature. (C) The portion of the transition curve used in van't Hoff analysis. (D) Plots of ΔH and ΔS as a function of temperature. (E) Thermal stability curve of hAR. (F) Triangular relationship among T_h, T_s, and ∆G_s. The explanation for T_g, T_g’, T_h, and T_s is given in the text. Dashed lines are extrapolations.The solid lines represent curves fitted to the 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 a global signal of change in the tertiary structure. ANS has been extensively used as a probe for non-native, partially unfolded conformations of the protein. The binding of ANS to hydrophobic regions results in a significant enhancement of ANS fluorescence and a pronounced blue-shift of the λ_max¹⁹.

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 a cooperative transition from native to unfolded state (Figure 2B and Figure 3B for urea and GuHCl, respectively). In the case of ANS fluorescence, a significant blue-shift of around 20 nm and 10 nm from the native to the 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 fluorescence) and (B2) λ_max (ANS fluorescence) against [urea]. (C1) I_max (Trp fluorescence) 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 (black), intermediate (cyan), and unfolded state (red). Solid lines represent curves fitted to the unfolding transitions; filled symbols represent data points from unfolding experiments; red symbols represent outliers.

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 (black), intermediate (cyan), and unfolded state (red). Solid lines represent curves fitted to the unfolding transitions; filled symbols represent data points from unfolding experiments; red symbols represent outliers.

A plot of I_max against denaturant concentration indicated the 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 based on the 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 the three-state model. In the case of ANS fluorescence, both I_max and fluorescence emission intensity at 480 nm fit equally well based on three-state model. Thus, Trp fluorescence emission intensity at 314 nm and ANS fluorescence emission intensity at 480 nm were analyzed based on the three-state model to evaluate the thermodynamic stability of hAR (Figure 2D and Figure 3D for urea and GuHCl, respectively). The thermodynamic parameters obtained from the fittings are listed in Table 1. Trp and ANS fluorescence demonstrate the 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).

Chemical induced unfolding monitored by far-UV CD

Unfolding profiles of hAR equilibrated at different denaturant concentrations monitored by far-UV CD are presented in Figure 4A1 and 4A2 for GuHCl and urea, respectively. The thermodynamic stability of hAR was determined based on the three-state model by plotting the 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. Far-UV CD profiles clearly distinguish three states (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 (black), intermediate (cyan), and unfolded state (red) for (C1) GuHCl and (C2) urea, respectively. The solid lines represent curves fitted to the unfolding transitions; filled symbols represent data points from unfolding experiments; red symbols represent outliers in data fitting.

Underlying data

Figshare: data_f1000_hAR_unfolding.zip. https://doi.org/10.6084/m9.figshare.8001998.v1¹⁸.

This project contains raw data for thermal denaturation and chemical 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).