GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle.
GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.
Miyazaki
Takuya
T
Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan.
Yoshimi
Tatsunari
T
Furutsu
Yoshinobu
Y
Hongo
Kunihiro
K
Mizobata
Tomohiro
T
Kanemori
Masaaki
M
Kawata
Yasushi
Y
D019943:Amino Acid Substitution; D000818:Animals; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism*; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D018833:Chaperonins / Q000378:metabolism*; D004798:Enzymes / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009876:Operon; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism*; D000516:alpha-Amylases / Q000378:metabolism
D018835:Chaperonin 10; D018834:Chaperonin 60; D004798:Enzymes; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013884:Thiosulfate Sulfurtransferase; D000516:alpha-Amylases; D018833:Chaperonins