Fig. 4

Co-expression of GroEL with PrkC and PrpC in E. coli and effect of phosphorylation on GroEL: a E. coli BL21 cells overexpressing His₆-GroEL with either PrkC_c or PrpC were metabolically labeled using [³²P]orthophosphoric acid. As shown in the autoradiogram, GroEL is phosphorylated in the presence of PrkC. Due to its affinity for GroEL, PrkC_c was also co-precipitated. b Purified His₆-GroEL co-expressed with either PrkC_c or PrpC were resolved by SDS-PAGE. The gel was stained with Pro-Q stain and analyzed by Typhoon imager. GroEL was phosphorylated when co-expressed with PrkC_c (GroEL-P), while co-expression with PrpC does not result in GroEL phosphorylation (GroEL-UP). c To understand the stoichiometry of phosphorylation of GroEL-P and GroEL-UP, the purified proteins (1 µg each) were separated by two-dimensional PAGE (pI range 4–7). The gels were immunoblotted on nitrocellulose membrane and developed using anti-GroEL antibodies. The images show respective autoradiograms of GroEL-UP (upper panel) and GroEL-P (lower panel). Multiple species of GroEL-P were observed indicating different levels of phosphorylation. d Gel filtration of purified GroEL-P and GroEL-UP. The graphs show presence of higher ratio of tetradecameric species in GroEL-P (upper panel) as compared with GroEL-UP (lower panel), which showed majority of dimer and heptamer. e Interaction of GroES with GroEL-P and GroEL-UP were analyzed by proteinase K-mediated partial cleavage. As compared with GroEL-UP:GroES, the phosphorylated GroEL-P:GroES complex was much more protected from protease cleavage indicating a stronger interaction. The experiment was performed thrice and the error bars show the SE of three independent readings