Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

Abstract

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

Keywords: Chaperone protein; Molybdenum cofactor; Molybdoenzymes; Thermofluor shift assay.

Figure 1

D1_anaerobic protein purity. Protein was expressed in DH5α from the pASG-IBA35 vector A. Steps of D1_anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1_anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1_anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).

Fig. 2

SEC HPLC-DAD analysis of D1_anaerobic, D1_aerobic and S25DH proteins. A. HPLC elution profile of D1_anaerobic (green – without DTT, orange – with DTT), D1_aerobic (grey – without DTT, yellow – with DTT) and S25DH (blue). For D1_aerobic (green line), three peaks were detected with masses corresponding to 29 kDa and 32 kDa, known as monomeric fractions (peak 3), and 59 kDa, known as dimer (peak 2). For D1_anaerobic (grey line), two peaks were detected; dimer (peak 2) and monomer (peak 3). In samples with DTT, there was only a single peak (peak 3) detected with a mass corresponding to 32 kDa (D1_anaerobic) or 35 kDa (D1_aerobic). B. Absorption spectra of compounds released from analyzed proteins (peak 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Comparison of Mo K-edge X-ray absorption near-edge spectra of the D1 chaperone (a) with Klebsiella pneumoneii nitrogenase MoFe protein (b), oxidized Rhodobacter sphaeroides DMSO reductase (c), and oxidized human sulfite oxidase (d). The vertical broken line is included to guide the eye to relative shifts of the spectra.

Figure 4

D1 chaperone Mo K-edge EXAFS (solid line) and best fit (broken line) (a) together with the corresponding EXAFS Fourier transforms (phase corrected for Mo—S backscattering) (b). EXAFS curve-fitting gives best fits with a mixed Mo—S/Mo—O first shell coordination with 3 Mo—S at 2.321(3) Å (σ²=0.0044(5)Ų) and 2 Mo—O at 2.21(5) Å (σ²=0.0031(7)Ų) with 2 Mo····Fe at 2.727(2) (σ²=0.0016(3)Ų). We note that 2 Mo····Cu at 2.717(2) (σ²=0.0025(1)Ų) gave an essentially equivalent fit but with a more chemically reasonable value for σ².

Figure 5

D1_aerobic protein purity. Protein was expressed in BL21(DE3)RILP from pMCSG7+D1 vector. A. Steps of D1_aerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution, 7 – D1_aerobic digested with rTEV, 8 – rTEV sample. B. The purity of D1_aerobic protein after SEC FPLC. This protein batch was used for TSA, ITC, crystallization and SEC FPLC analysis (lanes 1, 2 and 3).

Figure 6

SEC FPLC elution profile of D1_aerobic protein. A. Oligomerization of D1_aerobic after incubation in aerobic atmosphere. D1_aerobic forms oligomers in size of app. 110 kDa (black line) upon prolonged exposure to atmospheric oxygen. This process is reversible after addition of reducing agent (green line), the apparent size of D1_aerobic protein is 45 kDa). Normalized data of absorbance at 280 nm are presented.

Figure 7

No interaction between chaperone D1 and GDP was detected by thermofluor shift assay (TSA), size exclusion chromatography and isothermal titration calorimetry (ITC). A. Data from TSA experiments show that the stability of D1_aerobic is not affected by GDP. TSA was performed in a presence of decreasing GDP concentrations. T_m of D1_aerobic equaled 38 °C. The recorded change of D1_aerobic stability in the presence of GDP was less than 3 °C for each condition. B. Data from size exclusion chromatography. The absorbance at 280 nm of D1_aerobic protein did not change when GDP was added to the sample indicating that GDP does not interact with a protein. C. Data from ITC experiments of D1_aerobic with GDP. The plot presents integrated heat values of each injection. The blank values were subtracted from heat values generated after titration of GDP to D1_aerobic. No interaction between D1_aerobic and GDP was detected by ITC.

Fig. 8

Stability of D1_aerobic and D1_anaerobic measured by TSA. The numbers from 1 to 7 correspond to different buffers the compositions of which are presented in Table 1. The numbers above each chart correspond to the pH value of a given buffer. Green and orange plots represent recorded T_m values, while red and blue plots the dT_m = T_m (D1_anaerobic)-T_m (D1_aerobic.) (top) or dT_m = T_m (D1_anaerobic)-T_m (D1_anaerobic exposed to air) (bottom). A color scale for each type of plot is presented next to the plot. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

TSA measurements for ligands. Measurements were done in triplicate; an average value is shown. T_m for the D1 protein is 40°C. All tested compounds are listed in Table 2. Compounds for which it was not possible to calculate T_m or for which the dT_m was less than 3°C are not presented on the plot. The list of compounds for which T_ms was recorded is presented in Supplemental Fig. S5.