Folding and refolding of thermolabile and thermostable bacterial luciferases: the role of DnaKJ heat-shock proteins

Bacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loo se activity at 42 degrees C. Heat-denatured thermolabile Vibrio fischeri lu ciferase and thermostable Photo- rhabdus luminescens luciferase were used a s substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for d e novo folding at 30 degrees C of the luciferase, but essential for refoldi ng after a heat-shock. The rate and yield of DnaKJ refolding of the P. lumi nescens thermostable luciferase were to a marked degree lower as compared w ith the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37 de grees C, the refolding rates of the V. fischeri luciferase decreased and th e reaction reached a complete arrest at temperatures above 40 degrees C. Th e rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37 degrees C and then decreased at the range of 37 -44 degrees C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the ther molabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47 degrees C) temperature. The efficiency and reversibility of p rotein refolding arrest during and after heat-shock strongly depended on th e stability of the DnaKJ-denatured luciferase complex. It is supposed that the thermostable luciferase is released during the heat-shock, whereas the thermolabile luciferase remained bound to the chaperone. (C) 1999 Federatio n of European Biochemical Societies.