Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate

Proteins. 2011 Jun;79(6):1991-8. doi: 10.1002/prot.23024. Epub 2011 Apr 12.

Abstract

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Animals
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / metabolism*
  • Fireflies / genetics
  • Fireflies / metabolism
  • HSP40 Heat-Shock Proteins / metabolism*
  • HSP70 Heat-Shock Proteins / metabolism*
  • Heat-Shock Proteins / metabolism*
  • Hot Temperature
  • Luciferases / genetics
  • Luciferases / isolation & purification
  • Luciferases / metabolism*
  • Mutation
  • Protein Folding
  • Protein Stability

Substances

  • DnaJ protein, E coli
  • Escherichia coli Proteins
  • GrpE protein, E coli
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Luciferases
  • Adenosine Triphosphatases
  • dnaK protein, E coli