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

The role of chaperones Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA-ClpB-ClpX) in refolding of thermoinactivated luciferase from the marine bacterium Photobacterium fischeri and the terrestrial bacterium Photorhabdus luminescens has been studied. It is shown that refolding of thermoinactivated luciferases is completely determined by the DnaK-DnaJ-GrpE system. The degree of refolding of thermolabile 'quick' Ph. fischeri luciferase reaches 80% of the initial level over several minutes, whereas renaturation of thermostable 'slow' Ph. luminescens luciferase proceeds substantially slower (the degree of renaturation reaches only 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. luminescens luciferase revealed reduced thermostability in mutant strain E. coli clpA-. It was shown that this effect was not connected with DnaK-dependent refolding. These data suggest that denatured Ph. luminescens luciferase has enhanced affinity with respect to chaperone ClpA in comparison with DnaK, whereas thermolabile Ph. fischeri luciferase is characterized by enhanced affinity with respect to chaperone DnaK. Denatured luciferase bound to ClpA does not aggregate and following refolding proceeds probably spontaneously and very quickly (over 1-2 min). It is evident that the process under discussion requires ATP, since the addition of uncoupler of oxidative phosphorylation carbonyl cyanide 3-chlorophenylhydrazone results in a sharp decrease in thermal stability of luciferase to the level typical of the enzyme in vitro. The enhanced thermosensitivity of luciferases was observed also in E. coli containing mutations in gene clpB.