Bacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loose activity at 42³C. Heat-denatured thermolabile Vibrio fischeri luciferase and thermostable Photorhabdus luminescens luciferase were used as substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for de novo folding at 30³C of the luciferase, but essential for refolding after a heat-shock. The rate and yield of DnaKJ refolding of the P. luminescens thermostable luciferase were to a marked degree lower as compared with the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37³C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40³C. The rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37³C and then decreased at the range of 37^44³C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47³C) temperature. The efficiency and reversibility of protein refolding arrest during and after heatshock strongly depended on the 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. z 1999 Federation of European Biochemical Societies.
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