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 rapidly mixed with GroEL containing this mutation, and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Three kinetic phases were observed: a fast phase with a large amplitude and two slower phases with small amplitudes. The phases were assigned by (i) determining their dependence on ATP concentration; (ii) measuring their sensitivity to the mutation Arg197 --> Ala, which decreases cooperativity in ATP binding; and (iii) by carrying out mixing experiments of GroEL also with ADP, ATPgammaS, and ATP without K+. The apparent rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are strikingly similar to those determined in steady-state experiments. This phase, which reflects ATP-induced conformational changes, is sensitive to the mutation Arg197 --> Ala in a manner that parallels steady-state experiments. The rate of conformational change in the presence of ATP is >100 sec-1, which is fast relative to most protein folding rates, whereas in the absence of ATP it is approximately 0.7 s-1. The second phase reflects the transition from an ATP-bound state of GroEL 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) Biochemistry 34, 5303-5308].
Yifrach
O
O
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Horovitz
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
D018834:Chaperonin 60; D010649:Phenylalanine; D014364:Tryptophan; D000255:Adenosine Triphosphate