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J Biol Chem. 1996 Nov 8;271(45):28229-34.

GroEL locked in a closed conformation by an interdomain cross-link can bind ATP and polypeptide but cannot process further reaction steps.

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1
Tokyo Institute of Technology, Research Laboratory of Resources Utilization, R-1, 4259 Nagatsuta, Yokohama 226, Japan. myoshida@res.titech.ac.jp

Abstract

It has been believed that when GroEL binds to GroES its apical domain moves upward and outward. To inhibit this "opening" movement, its equatorial and apical domains were cross-linked through a disulfide bond between mutationally introduced cysteine residues at the positions of Asp-83 and Lys-327. To avoid possible undesired cross-linking, we at first prepared a mutant GroEL (GroELNC; Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser) in which all cysteine residues in wild-type GroEL were replaced by serine residues. GroELNC was fully functional as a chaperonin. We then introduced the above two point mutations into GroELNC to generate a mutant (GroELAEX; Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser and Asp-83 --> Cys, Lys-327 --> Cys). Oxidized GroELAEX, which is locked in a "closed" conformation by an interdomain disulfide bond, can bind 6-7 mol of ATP, which remain bound without hydrolysis. This ATP-bound, oxidized GroELAEX can bind the stably nonnative substrate protein isopropylmalate dehydrogenase, whereas the nucleotide-free oxidized GroELAEX binds it with a weaker affinity. However, oxidized GroELAEX fails to process further reaction steps such as ATP hydrolysis, binding of GroES, dissociation of substrate protein from GroEL, and facilitating protein folding. When disulfide bonds in oxidized GroELAEX are reduced, GroELAEX exerts the ability to process all the reactions just as GroELNC and wild-type GroEL. Indications from these results are: hydrolysis of ATP may require opening movement of the apical domain; GroES binds to an open form of GroEL; and substrate polypeptide is released from GroEL coupled with either ATP hydrolysis or opening movement of the apical domain.

PMID:
8910440
[Indexed for MEDLINE]
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