Overproduction of proteins was performed in E. coli BL21(DE3) RIL in TB medium with 5 M FAD. The wild-type MetF protein was purified as described previously (Sheppard et al. 1999). The 117 variant of MetF was purified analogously as the wild-type enzyme, but all buffers used for purification of MetF117 were supplemented with 5 μM FAD.
DnaK, GrpE, DnaJ, ClpB and HtpG were overproduced and purified according to the previously published methods (Banecki and Zylicz 1996; Banecki et al. 1996; Spence and Georgopoulos 1989; Woo et al. 1992; Wawrzynów et al. 1995).
Determination of methylenetetrahydrofolate reductase activity
Determination of methylenetetrahydrofolate reductase was performed as described previously (Sheppard et al. 1999), by measuring a decrease in absorbance of NADH, consumed during the reaction. The reaction mixture consisted of 50 mM phosphate buffer containing 10 % glycerol, 0.3 mM EDTA, 400 μM NADH, and 1.4 mM menadione (vitamin K3 was used as an artificial substrate for MetF). The activity of MetF was determined by measurement of the kinetics of the reaction at 37 °C. The reaction mixture was prepared without the enzyme, and incubated for 5 min. Following reaction initiation by the addition of 0.3 μM enzyme, the measurement was carried out for 30 min, by monitoring the absorbance at a wavelength of 340 nm.
Hsps protection of MetF117 from temperature-mediated denaturation
MetF (at final concentration of 0.3 μM) was pre-incubated in the presence of 50 mM phosphate buffer (pH 7.2) containing 10 % glycerol, 0.3 mM EDTA, 50 mM NaCl, 20 mM KCl, 20 mM MgCl2, and in the presence of DnaK, DnaJ, GrpE, ClpB (the Hsp 70/100 system) or HtpG, and 5 mM ATP. The concentrations of heat shock proteins were 3.7 μM, 1.4 μM, 0.36 μM, 1.5 μM, respectively for Hsp70/100, and 3.6 μM for HtpG. The incubation was carried out for 15 min at 50 °C. Finally, the samples were subjected to the MetF activity test.
Reactivation of thermally-denatured MetF117 by Hsps
MetF (at final concentration of 0.3 μM) was thermally inactivated by 15 min incubation at 50 °C in the presence of 50 mM phosphate buffer (pH = 7.2) containing 10 % glycerol, 0.3 mM EDTA, 50 mM NaCl, 20 mM KCl, and 20 mM MgCl2. Following the incubation, the enzyme was renatured in the presence of Hsp 70/100 or HtpG at the concentrations indicated in the preceding subsection. The renaturation was carried out for 45 min at 20 °C. Finally, the samples were subjected to the MetF activity test.
Statistical analysis was performed using T-test. A p value < 0.05 was considered to indicate statistical significance. Each experiment was repeated three times. All data were calculated with Statistica 12 software (StatSoft).