Abstract
The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.
Highlights
The chaperonin GroEL (14-mer) from Escherichia coli binds denatured proteins and facilitates their folding in vivo and in vitro by encapsulating them within an isolated cavity formed in cooperation with the co-chaperonin GroES (7-mer) [1, 2]
These results indicated that the C-terminal region of chaperonin is involved in multiple facets of the chaperonin mechanism, i.e. in ATP hydrolysis activity, stabilization of chaperonin quaternary structure, and substrate protein folding
To understand how the C-terminal region contributes to the cavity environment, we examined the hydrophobicity of all truncated mutants, influenced by previous studies on the C-terminal region of Hsp60 homologue from S. cerevisiae, which showed that the predominantly hydrophobic C terminus of Hsp60 is a very important factor for proper protein folding inside the central cavity [17]
Summary
Reported no acceleration in folding rate in these tail-elongated single-ring GroEL mutants. We focused on the final 23 amino acid residues (526KNDAADLGAAGGMGGMGGMGGMM548) at the C-terminal region of E. coli GroEL, which are invisible in x-ray crystal structures, and attempted to understand the role of this region in chaperonin function. The critical importance of the hydrophilic nature of this region for chaperonin function was confirmed by modifying this sequence in a controlled manner These results showed clearly that the hydrophilic nature of 526KNDAAD531 is critical in maintaining a proper environment for substrate protein folding within the central cavity. This is the first report that identifies specific amino acid residues within the crystallographically disordered C-terminal region that are critical to chaperonin function
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