FIGURE 4.

Dimer mutations R56A, T301A, N537A, and D540A compromise the formation of DnaK dimer. A, native gel analysis of the dimer mutations in the DnaK-T199A/L′3,4 construct. 2 μl of each protein at 12 mg/ml was loaded onto an 8–25% gradient PhastGel (GE Healthcare). The positions of the monomer (1×) and dimer (2×) are indicated by arrows. The DnaK-T199A/L′3,4 protein (WT) was used as control. B, all the dimer mutants in the DnaK-T199A/L′3,4 construct were purified to a similar purity as that of the DnaK-T199A/L′3,4 (WT). 24 μg of each protein were loaded onto SDS-PAGE. C and D, AUC analysis of the dimer mutants in the presence of ATP (C) and ADP (D), respectively. Each mutant protein (at 1 mg/ml) was subjected to sedimentation velocity experiment as in Fig. 2D. The WT DnaK was used as control. The positions of the monomer and dimer are labeled as 1× and 2×, respectively. E, glutaraldehyde cross-linking. The mutant DnaK proteins were treated with 0.00625, 0.0125, and 0.025% glutaraldehyde and separated on SDS-PAGE as in Fig. 2F. The concentrations of glutaraldehyde were labeled at the top of the gel. The positions of the monomer (1×) and dimer (2×) are labeled on the right. F, dimer mutants T301A and N537A/D540A compromise the formation of the disulfide bond between A303C and H541C. T301A and N537A/D540A were introduced into the cysteine mutants A303C and H541C. Purified proteins were oxidized with increasing concentrations of copper-phenanthroline (50, 100, and 200 μm; labeled at the top of the gel) in the presence of ATP as described in Fig. 2G. The positions of the monomer (1×) and dimer (2×) are labeled on the right. G, cysteine mutant DnaK proteins used in F were purified to the similar purity as that of the WT DnaK. Each DnaK protein was loaded and separated on an SDS-polyacrylamide gel.