Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATPanalogs 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 A TP binding or hydrolysis. One of the main concerns of GroEL is the mechanis m of the productive and cooperative structural change of GroEL induced by t he nucleotide. We studied the cooperative nature of GroEL by nucleotide tit ration 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 tha t GroEL has two kinds of nucleotide binding sites. The fluorescence titrati on result fits well with a model in which two kinds of binding sites are bo th non-cooperative and independent of each other. These results suggest tha t the binding and hydrolysis of ATP may be necessary for the cooperative tr ansition of GroEL. (C) 2001 Elsevier Science B.V. All rights reserved.