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

G. B. Zavilgelsky^1*, V. Yu. Kotova¹, M. M. Mazhul'², and I. V. Manukhov¹

¹State Scientific Center of the Russian Federation GosNIIgenetika, 1-yi Dorozhnyi Proezd 1, Moscow, 117545 Russia; fax: (095) 315-0501; E-mail: zavilgel@genetika.ru

²Department of Microbiology, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia

^* To whom correspondence should be addressed.

Received April 5, 2002; Revision received April 17, 2002

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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. 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 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. In the case of thermolabile Ph. fischeri luciferase, mutation in gene clpA has no effect on the shape of the curve of thermal inactivation. 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. 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).

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KEY WORDS: chaperones, DnaK, ClpA, ClpB, refolding, luciferase

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