DnaK/DnaJ chaperone system reactivates endogenous E. coli thermostable FBP aldolase in vivo and in vitro; the effect is enhanced by GroE heat shock proteins.
Thermally aggregated, endogenous proteins in Escherichia coli cells form the S fraction, which is separable by sucrose density gradient centrifugation. To date, relatively little is known about the mechanisms of elimination of the heat-aggregated proteins from E. coli cells and the composition of the S fraction. We have identified several proteins of the S fraction using 2D-gel electrophoresis and microsequencing. A thermostable II class fructose-1,6-bisphosphate aldolase (Fda protein) appeared to be one of numerous proteins of the S fraction. Fda was purified from E. coli overproducer strain and used as a model substrate for investigation of the role of Hsps in prevention and repair of thermal denaturation of proteins both in vivo and in vitro. We found that the heat inactivation of Fda was reversible and that its reactivation in vivo and in vitro required mainly the assistance of the DnaK/DnaJ chaperone system. The dnaK756 and dnaJ259 mutations had a negative effect on the reactivation of thermally inactivated Fda. Moreover, we showed that the reactivation process in vitro was enhanced when GroEL/GroES were added together with DnaK/DnaJ. GroEL/GroES alone were inefficient in the resolubilization or reactivation of the heat-aggregated Fda. It is supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of Hsps than from the direct heat effect.
Kedzierska
S
S
Department of Biochemistry, University of Gdańsk, Poland. kedzie@biotech.univ.gda.pl
Jezierski
G
G
Taylor
A
A
D002458:Cell Fractionation; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D066298:In Vitro Techniques; D018832:Molecular Chaperones / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D011489:Protein Denaturation
D018835:Chaperonin 10; D018834:Chaperonin 60; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C063842:dnaK protein, E coli; D005634:Fructose-Bisphosphate Aldolase