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     The atomic-resolution structures of the relatively stable (T and R) end states of several allosteric proteins are known but the pathways by which they interconvert are generally not known. We are trying to address this issue using GroEL as a model system. One approach is to employ linear free energy relationships of physical organic chemistry which have been found very useful in analysing protein folding reactions. We have begun to characterize the transition state of the T to R reaction of GroEL using Bronsted analysis (Yifrach and Horovitz, 1998). Our data so far suggests that in the transition state the inter-subunit R197-E386 salt-link is broken thus enabling rotation of subunits in the plane of the ring but that the upward shift of the apical domains has not yet taken place. Our data also led to deriving a kinetic criterion for concerted allosteric transitions (Horovitz and Yifrach, 2000). In addition, we are characterizing potentially important interactions in the R state of GroEL (for which there is no available atomic-resolution structure) using double-mutant cycles (Horovitz, 1996; Aharoni and Horovitz, 1997). We are also interested in theoretical methods that may shed light on pathways of information transfer in this molecule such as analysis of correlated mutations (Kass and Horovitz, 2002; Noivirt et al., 2005). In connection with this research question, we have also recently begun cryo-EM analysis of the R13G, A126V mutant of GroEL whose crystal structures in the apo and ATPgammaS-bound states have been determined. This mutant is defective in inter-ring allostery (Aharoni and Horovitz, 1996) and by solving the structure we hope to gain insight into the structural basis of negative allostery in GroEL which remains poorly understood. This project is in collaboration with Dr. Sharon Wolf from the W.I.S. and Prof. Helen Saibil from Birkbeck College, London.