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

Sandeep K. Sharma, Paolo De Los Rios, Pierre Goloubinoff*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Scopus citations


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.

Original languageEnglish
Pages (from-to)1991-1998
Number of pages8
JournalProteins: Structure, Function and Bioinformatics
Issue number6
StatePublished - Jun 2011
Externally publishedYes


  • Chaperone
  • DnaJ
  • DnaK
  • GrpE
  • Luciferase
  • Misfolding
  • Protein aggregation
  • Thioflavin-T
  • Unfolding


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