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 loose activity at 42 degrees C. 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 30 degrees C 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 37 degrees C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40 degrees C. The 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 thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47 degrees C) temperature. The efficiency and reversibility of protein refolding arrest during and after heat-shock 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.
Manukhov
I V
IV
State Scientific Centre of Russian Federation GNIIGENETIKA, Moscow.
Eroshnikov
G E
GE
Vyssokikh
M Y
MY
Zavilgelsky
G B
GB
D001426:Bacterial Proteins; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D010957:Plasmids; D017510:Protein Folding; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008156:Luciferases; C063842:dnaK protein, E coli