TRANSIENT KINETIC-ANALYSIS OF ADENOSINE 5'-TRIPHOSPHATE BINDING-INDUCED CONFORMATIONAL-CHANGES IN THE ALLOSTERIC CHAPERONIN GROEL

GroEL with an intrinsic fluorescent probe was generated by introducing the mutation Phe44 --> Trp. Different concentrations of ATP were rapi dly mixed with GroEL containing this mutation, and the time-resolved c hange in fluorescence emission, upon excitation at 280 nm, was followe d. Three kinetic phases were observed: a fast phase with a large ampli tude and two slower phases with small amplitudes. The phases were assi gned by (i) determining their dependence on ATP concentration; (ii) me asuring their sensitivity to the mutation Arg197 --> Ala, which decrea ses cooperativity in ATP binding; and (iii) by carrying out mixing exp eriments of GroEL also with ADP, ATP gamma S, and ATP without K+. The apparent rate constant corresponding to the fast phase displays a bi-s igmoidal dependence on ATP concentration with Hill coefficients that a re strikingly similar to those determined in steady-state experiments. This phase, which reflects ATP-induced conformational changes, is sen sitive to the mutation Arg197 --> Ala in a manner that parallels stead y-state experiments. The rate of conformational change in the presence of ATP is > 100 sec(-1), which is fast relative to most protein foldi ng rates, whereas in the absence of ATP it is similar to 0.7 s(-1). Th e second phase reflects the transition from an ATP-bound state of GroE L to an ADP-bound state. The third phase, with the smallest amplitude, reflects release of residual contaminants. The results in this study are found to be consistent with the nested model for cooperativity in ATP binding by GroEL [Yifrach, O., and Horovitz, A. (1995) Biochemistr y 34, 5303-5308].