Abstract
To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by approximately 2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of approximately 3- and approximately 5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434 g). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an approximately 5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state.
Highlights
To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed
We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an ϳ5-s duration
The substrate protein is encapsulated in the GroEL cavity underneath GroES, where it folds during the time of ATP hydrolysis [1, 2]
Summary
We constructed a C-terminal truncated mutant (termed ELtc20), which lacks the final 20 amino acid residues, and characterized it. ELtc was shown to have the basic functions of GroEL and the extended functional cycle (ϳ10 s) in comparison with that of the wild type (ϳ8 s). We proposed a model for the functional cycle of GroEL with two successive rate-limiting steps of ϳ3 and ϳ5 s duration [17]. Detailed kinetic studies revealed that the C-terminal truncation extends the lifetime of the first rate-limiting step to ϳ5 s. From these results, we suggest the notion that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state in the functional cycle
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