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 526 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). Encapsulation by GroEL protects the denatured proteins from interactions with other misfolded proteins or aggregation prone species, providing the proper environment in which the denatured protein may fold spontaneously (3-6). The unique quaternary structure (two heptameric rings stacked back to back) of GroEL enables a clever mechanism. The subunit structure (548 amino acid residues) is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The apical domain plays an important role in recognizing and binding denatured protein and the co-chaperonin GroES. The intermediate domain connects the apical and the equatorial domains, and the equatorial domain binds ATP and hydrolyzes it. This ATP hydrolysis controls the overall chaperonin mechanism, regulating binding and release of the substrate protein and GroES (7-11). The refolding substrate protein is encapsulated by GroEL-GroES and segregated from the surrounding environment and, under these conditions, folds correctly without forming aggregation.Thus, the mechanism of GroEL-mediated protein folding is well characterized. However, some details regarding the specific roles of various structural elements in the GroEL subunit structure remain unclear. For example, as shown in Fig. 1, the final 23 amino acid residues of the C terminus are not clearly defined in x-ray crystallographic studies (9) due to high flexibility. However, these segments of GroEL oligomer appear to coalesce and block the central channel at the level of the equatorial domain in electron micrograph (12) and small-angle neutron scattering (13) experiments. Previously, it was reported that this C-terminal region is closely involved in the rate of ATP hydrolysis (14) and GroEL oligomerization (15, 16). However, a mutant with 27-amino acid residues truncated from the C terminus was still functional and could support normal growth of the host cell (15,16). Moreover, the role of the C-terminal region in the mitochondrial homologue Hsp60 (572 amino acid residues) from Saccharo...

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