Role of Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA and ClpB) Chaperones in Refolding and Increased Thermal Stability of Bacterial Luciferases in Escherichia coli Cells

摘要

The role of chaperones Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA-ClpB-ClpX) in refolding of thermoin-actvated luciferase from the marine bacterium Photobacterium fischeri and the terrestrial bacterium Photorhabdus luminescens has been studied. These luciferases are homologous, but differ greatly in the rate of thermal inactivation and the rate constant for the luminescence reaction. It was shown that refolding of thermoinactivated luciferases is completely determined by the DnaK-DnaJ-GrpE system. However these luciferases markedly differ in the rate and degree of refolding. The degree of refolding of thermostable "slow" Ph. luminescens luciferase proceeds substantially slower (the degree of renaturation reaches only approx 7-8% of the initial level over tens of minutes). The measurement of the rate of thermal inactivation of luciferases in vivo in the cells of Escherichia coli wild strain and strains containing mutations in genes clpA, clpB, clpX showed that Ph. lumi-nescens luciferase revealed reduced thermostability in mutant strain E. coli clpA~-. It was shown that this effect was not con-nected with DnaK-dependent refolding. In the case of thermal inactivation. These data suggest that denatured Ph. luminescens luciferase has enhanced affinity with respect to chaperone ClpA in comparision with DnaK, whereas thermolabile Ph. fischeri luciferase is character-ized by enhanced affinity with respect to chaperone DnaK. Denatured luciferase bound to ClpA does not aggregate under dis-cussion requires ATP, since the addition of uncoupler of oxidative phosphorylation carbonyl cyanide 3-chlorophenylhydra-zone results in a sharp decrease in thermal stability of luciferase to the level typical of the enzyme in vitro. The enhanced ther-mosensitivity of lucifierases was observed also in E. coli containing mutations in gene clpB. However, this effect, which takes place for Ph. fischeri luciferase as well as for Ph. luminescens luciferase, is determined by DnaK-dependent refolding and probably connected with the ability of chaperone ClpB to provide disaggregation of the proteins, resulting in their interaction with chaperones of the Hsp70 family (DnaK-DnaJ-GrpE).