The substrate binding domain of DnaK facilitates slow protein refolding.
We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of beta-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active beta-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of beta-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.
Tanaka
Naoki
N
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Japan. tanaka@ipc.kit.ac.jp
Nakao
Shota
S
Wadai
Hiromasa
H
Ikeda
Shoichi
S
Chatellier
Jean
J
Kunugi
Shigeru
S
D001665:Binding Sites; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D001616:beta-Galactosidase; C063842:dnaK protein, E coli