Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL

Journal of Molecular Biology
G P TsurupaK Kuwajima

Abstract

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 we...Continue Reading

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Citations

Jun 1, 2002·Journal of Biological Physics·K KuwajimaT Inobe
Nov 19, 2013·PLoS Computational Biology·Murat Cetinbaş, Eugene I Shakhnovich
Apr 20, 2007·Cell Stress & Chaperones·Kristin N Parent, Carolyn M Teschke
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