Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL.
We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements. It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1. The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution. In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme. On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1. The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions. Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding. The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP.
Tsurupa
G P
GP
School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113, Bunkyo-ku, Japan.
Ikura
T
T
Makio
T
T
Kuwajima
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology; D018835:Chaperonin 10 / Q000494:pharmacology; D018834:Chaperonin 60 / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009994:Osmolar Concentration; D017510:Protein Folding
D018835:Chaperonin 10; D018834:Chaperonin 60; D000255:Adenosine Triphosphate; D008836:Micrococcal Nuclease