Kinetic analysis of ATP-dependent inter-ring communication in GroEL

Different concentrations of ATP were mixed rapidly with single-ring or double-ring forms of GroEL containing the Phe44 --> Trp mutation and the time-resolved changes in fluorescence emission, upon excitation at 295 nm, were followed. Two kinetic phases that were previously found for double-ring GroEL are also observed in the case of the single-ring version: (i) a fast phase with a relatively large amplitude that is associated with the T --> R allosteric transition; (ii) and a slow phase with a smaller amplitude that is associated with ATP hydrolysis. In the case of weak intra-ring positive cooperativity, the rate constant corresponding to the T --> R allosteric switch of single-ring GroEL displays a bi-sigmoidal dependence on ATP concentration that may reflect parallel pathways of the allosteric transition. The slow phase is absent when double-ring or single-ring forms of GroEL are mixed with ADP or ATP without K+, and it has a rate constant that is independent of ATP concentration. A third fast phase that is still unassigned is observed for both single-ring and double-ring GroEL when a large amount of data is collected. Finally, a fourth phase is observed in the case of double-ring GroEL that is found to be absent in the case of single-ring GroEL. This phase is here assigned to inter-ring communication by (i) determining its dependence on ATP concentration and (ii) based on its absence from single-ring GroEL and the Arg13 --> Gly, Ala126 --> Val GroEL mutant, which is defective in inter-ring negative cooperativity. The value of the rate constant corresponding to this phase is found to increase with increasing intra-ring positive cooperativity, with respect to ATP. This is the first report of the rate of ATP-mediated inter-ring communication in GroEL, in the presence of ATP alone, which is crucial for the cycling of this molecular machine between different functional states. (C) 2004 Elsevier Ltd. All rights reserved.