Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP.
Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.
Inobe
T
T
Department of Physics, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan.
Makio
T
T
Takasu-Ishikawa
E
E
Terada
T P
TP
Kuwajima
K
K
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site; D001665:Binding Sites; D002151:Calorimetry; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D008301:Maleimides; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence
D000227:Adenine Nucleotides; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D008301:Maleimides; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C003944:N-(3-pyrene)maleimide; D000251:Adenosine Triphosphatases