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 k inetics of staphylococcal nuclease and its three mutants by stopped-fl ow fluorescence measurements. It was found that a transient folding in termediate of staphylococcal nuclease was tightly bound to GroEL and r efolded in the Groin-bound state without releasing the non-native prot ein in solution, and the refolding rate in the Groin-bound state was 0 .01 s(-1) The GroEL-affected refolding of the nuclease appears to be i n decided contrast to that of apo-alpha-lactalbumin reported in our pr evious study, wherein alpha-lactalbumin was shown to be more weakly bo und by GroEL and to refold in the free state in solution. Ln spite of the apparent difference between the proteins, the Groin-affected refol ding reactions of both the proteins can be represented by a common uni fied reaction scheme. On the basis of this scheme, the binding constan t between the nuclease intermediate and Groin was estimated to be larg er than 10(9) M-1. The stoichiometry of binding of the nuclease and it s mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The in crease in ionic strength resulted in a weakening of the interaction be tween the nuclease and GroEL, which was attributed to a weakening of t he electrostatic attraction between the two proteins as a result of el ectrostatic screening by ions. Although ATP was found to accelerate th e GroEL-affected refolding of the nuclease, the refolding rate was sti ll far from the rate of the free refolding. The free refolding behavio r of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP. (C) 1998 Academic Press Limited.