Figure 1.
Positions of mutations in the primary and tertiary structures of GroEL.
(A) Alignment of the amino acid sequences of GroEL and the mature mHsp60 protein. Protein sequence alignments were carried out by ClustalW. Amino acids discussed in this study are marked in boldface type. The amino acids known to be in direct contact with GroES are underlined. The color code corresponding to domain boundaries is described below. (B–C) 3D-structure models of GroEL subunit in the down (B) and up (C) conformations (Protein Data Bank entry 1AON) [43]. The amino acids discussed in this study are labeled and presented as space-filling models. The corresponding amino acids in mHsp60 are indicated in brackets. The ADP molecule is colored in purple. The three domains as defined by GroEL are color-coded on the GroEL sequence and structure: equatorial (blue), intermediate (green) and apical (red). Helices H and I are colored in gray. The figure was produced using PyMOL software.
Figure 2.
Identifying mHsp60 mutants that are functional with GroES.
(A) Examination of the in vivo system at the indicated growth conditions. (B) Ten-fold-serial dilutions of E. coli strain MGM100 harboring plasmid pOFX with the indicated mHsp60 variant and GroES, grown on agar plates in the presence of glucose and IPTG. (C–D) Examination of the ability of mHsp60 mutants to facilitate the growth of MGM100 on agar plates containing glucose and IPTG in combination with GroES (C) or mHsp10 (D). GroEL-GroES and mHsp60-mHsp10 combinations serve as positive controls; the mHsp60-GroES combination serves as negative control. (E) Refolding of 0.33 µM HCl-denatured MDH by 10 µM of the indicated chaperonin and 40 µM of mHsp10 (white columns) or GroES (black columns). MDH activity was measured at 340 nm following 120 min incubation at 30°C in the presence of 1 mM ATP. The activity following refolding is presented relative to that of native MDH (100%).
Figure 3.
Inhibition of GroES-E321K refolding activity by mHsp10.
A binary complex of E321K and HCl-denaturated MDH was pre-incubated for 30 min in the presence of increasing concentrations (from 0 to 20 µM) of mHsp10 and 2 mM ATP before adding 20 µM GroES. MDH activity was measured 1 hour following the addition of GroES. % inhibition = 100*[(Ao–Ai)/Ao]. Ao represents the activity level in the absence of mHsp10, and Ai represents the activity level at each mHsp10 concentration.
Figure 4.
A stable complex is formed between the E321K mutant and mHsp10.
Interaction between mHsp10 and different chaperonins was measured using a pulldown assay. 50 µM of His-tagged mHsp10 together with 50 µM of GroEL (A), mHsp60 (B), or E321K mutant (C) were incubated with nickel beads in the absence of nucleotides, or in the presence of 4 mM ATP or 4 mM ADP. Equivalent aliquots of 2 µl from the total sample (T), unbound fraction (U), fourth wash (W), and bound fraction (B) were analyzed by SDS-PAGE and stained with Coomassie blue. The intensities of the bands were quantified by densitometry (ImageMaster 1D Prime program). The bound ratio listed on the bottom of each gel represents the ratio between the intensities of the chaperonin and co-chaperonin bands in the bound fraction.
Figure 5.
Refolding activity in the presence of increasing concentrations of wild-type and low-affinity co-chaperonins.
Refolding of 0.33 µM HCl-denatured MDH by 10 µM of chaperonin GroEL (A), wild-type mHsp60 (B), E321K mHsp60 (C), R264K/E358K mHsp60 (D), in the presence of increasing concentrations of mHsp10 (white triangles), GroES (black triangles) and the low-affinity mutants: mHsp10_L33A (white diamonds) and GroES_L27A (black diamonds). MDH activity was measured at 340 nm following 120 min incubation at 30°C in the presence of 1 mM ATP. The 100% reference was determined as the activity of a sample containing the same amount of native MDH.
Table 1.
Inhibition of chaperonin ATPase activity by various co-chaperonins.
Figure 6.
Chaperonin-co-chaperonin interactions measured by SPR.
Association and dissociation patterns of 10 µM of the indicated chaperonin to immobilized (A) GroES (∼ 600 Relative Units-RU) or (B) mHsp10 (∼ 800 RU) in the presence of 2 mM ATP.
Table 2.
SPR analysis.
Figure 7.
The effect of the K176E mutation on the function of mHsp60 with co-chaperonins.
Refolding of 0.33 µM HCl-denatured MDH by 10 µM of the indicated chaperonin and 20 µM of mHsp10 (white columns) or GroES (black columns). MDH activity was measured at 340 nm following 60 min incubation at 30 °C in the presence of 1 mM ATP. The activity following refolding is presented relative to that of native MDH (100%).
Figure 8.
The inhibitory effect of ADP on MDH refolding activity by chaperonins.
(A–D) Refolding of 0.33 µM HCl-denatured MDH by 10 µM of the indicated chaperonin and 40 µM of mHsp10 (white columns) or GroES (black columns). MDH activity was measured at 340 nm following a 60 min incubation at 30°C in the absence of nucleotides or in the presence of 10 mM ADP, 1 mM ATP or 1 mM ATP+10 mM ADP as indicated. The activity following refolding is presented relative to that of native MDH (100%). (E–G) Time-dependent refolding activity of wild-type mHsp60 (E), E321K mutant (F) and R264K/E358K mutant (G) together with mHsp10 (white symbols) or GroES (black symbols) in the presence of 1 mM ATP (triangles) or 1 mM ATP+10 mM ADP (squares). The relative activity is compared to the activity measured by each chaperonin pair after 30 min in the presence of ATP (100%).