Preformed GroES oligomers are not required as functional cochaperonins.
We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268-30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96-97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer-monomer equilibrium to be 7.3 x 10(-34)M6. The GroESD(96-97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96-97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the performed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.
Seale
J W
JW
Department of Biochemistry, University of Texas Health Sciences Center at San Antonio 78240-7760, USA.
Chirgwin
J M
JM
Demeler
B
B
Horowitz
P M
PM
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D018835:Chaperonin 10 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D018834:Chaperonin 60 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D046911:Macromolecular Substances; D016296:Mutagenesis; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry; D014461:Ultracentrifugation; D014508:Urea
D018835:Chaperonin 10; D018834:Chaperonin 60; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D000251:Adenosine Triphosphatases