Folding of a misfolding-prone beta-galactosidase in absence of DnaK.
In absence of chaperone DnaK, bacterially produced misfolding-prone proteins aggregate into large inclusion bodies, but still a significant part of these polypeptides remains in the soluble cell fraction. The functional analysis of the model beta-galactosidase fusion protein VP1LAC produced in DnaK(-) cells has revealed that the soluble version exhibits important folding defects and that it is less stable and less active than when produced in wild-type DnaK(+) cells. In addition, we have observed that the induction of gene expression at the very late exponential phase enhances twofold the stability of VP1LAC, a fact that in DnaK(-) background results in a dramatic increase of its specific activity up to phenotypically detectable levels. These results indicate that the chaperone DnaK is critical for the folding of misfolding-prone proteins and also that the soluble form reached in its absence by a fraction of polypeptides is not necessarily supportive of biological activity. In the case of E. coli beta-galactosidase, the catalytic activity requires assembling into tetramers and the fine organization of the activating interfaces holding the active sites, what might not be properly reached in absence of DnaK.
García-Fruitós
Elena
E
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Carrió
M Mar
MM
Arís
Anna
A
Villaverde
Antonio
A
D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000172:deficiency* / Q000235:genetics; D018869:Heat-Shock Response / Q000502:physiology*; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D001616:beta-Galactosidase; C063842:dnaK protein, E coli