FIG 8 .
In vitro proteolysis of the monomeric and dimeric forms of SmcR by ClpPA and Lon. (A) Monomeric and dimeric forms of recombinant SmcR. The original SmcR (SmcRWT) and mutated SmcR (SmcRY171A/C198A) were incubated in the presence (+DTT) or absence (−DTT) of 1 mM DTT and fractionated by SDS-PAGE. The protein bands were visualized by Coomassie blue staining. Monomeric and dimeric forms are designated with arrows. (B) Secondary structure assay of rSmcR. Changes in the secondary structure of SmcRWT and SmcRY171A/C198A were monitored by scanning each recombinant protein (2.3 mg/ml) at 200 to 250 nm. The mean ellipticity of SmcRWT and SmcRY171A/C198A was recorded and is indicated with solid and dotted lines, respectively. (C) Thermal stability assay of rSmcR. Thermal denaturation profiles of SmcRWT and SmcRY171A/C198A (0.23 mg/ml each) were monitored by temperature-dependent CD. The change of ellipticity at 222 nm was used to calculate the percentages of unfolding protein, and the results are plotted with solid (SmcRWT) and dotted (SmcRY171A/C198A) lines. (D) Time series proteolysis of monomeric and dimeric SmcR by ClpPA. SmcRWT and SmcRY171A/C198A (2.0 μM each) were incubated with rClpP (2.2 μM) and rClpA (0.6 μM) in the absence (the left gels) or presence (the right gels) of 1 mM DTT. At various time points, the proteolysis reaction was stopped by adding a loading buffer and boiling. The resulting reaction mixtures were separated by SDS-PAGE and visualized by Coomassie blue staining. The levels of remaining rSmcR proteins were estimated by densitometric reading of the corresponding bands, and the relative levels of abundance compared with the rSmcR in the absence of ClpPA (lanes 2) were plotted against incubation time. (E) Time series proteolysis of monomeric and dimeric SmcR by Lon. SmcRWT and SmcRY171A/C198A (2.0 μM each) were incubated with rLon (0.6 μM) in the absence (the left gels) or presence (the right gels) of 1 mM DTT. Proteolysis results are presented as described above.