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

Abstract

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.

Reversible Thermal Transition in GrpE, the Nucleotide Exchange Factor of the DnaK Heat-Shock System

“…of the T 3 R conversion, observed in the presence of a protein substrate (11), the shift might result in the sequestering of protein substrates at heat-shock temperatures. During heat shock, DnaK and DnaJ have indeed been reported to cooperatively retain thermally unfolded substrate protein in a folding competent state both in vivo (31,32) and in vitro (31,33), whereas GrpE is required for the reactivation of the substrate protein after the heat shock (31,33). The occurrence of a sequestering mechanism at heat-shock temperatures does not preclude the possibility of an additional mechanism of action in which DnaK uses the energy of ATP hydrolysis to exert conformational work upon polypeptide substrates that have undergone off-pathway folding (10,34).…”

“…Obviously, the balance of ATP hydrolysis and nucleotide exchange, accelerated by DnaJ and GrpE, respectively, and thus the ratio of T-state to R-state DnaK, are important for effective refolding of denatured proteins. The amount of the components of the DnaK heat-shock system is known to be controlled through the regulation of transcription, stability, and activity of 32 (for a review, see Ref. 12).…”

“…The expression of DnaK and the co-chaperones DnaJ and GrpE is controlled by the transcription factor 32 (for a review, see Ref. 12).…”

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

“…The thermostabilities of bacterial luciferases are known to vary depending on the organism in which luciferase is expressed (13). Furthermore, V. fischeri luciferase is thermolabile, with enzyme denaturation occurring above 30°C (14). In addition, a similar result was observed during naphthalene catabolism since peak luminescence was highest at pH 6.0, a value below the pH of 7.0 that is optimum for naphthalene dioxygenase activity (Fig.…”

Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems

“…AR effects on the process of chaperone independent refolding of bacterial luciferase were studied using a [15]. The bacteria were grown at 28°C with forced aeration for 3 h in 15 mL flasks containing 5 mL LB broth (Sigma) in the pres ence of 100 μg/mL ampicillin to the early exponential growth phase (OD = 0.2, λ = 640 nm; Specord, Ger many).…”