title: Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate.
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.
authors.affiliation: Département de Biologie Moléculaire Végétale, Université de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Sandeep K
authors.initials: SK
authors.lastname: Sharma
authors.forename: Paolo
authors.initials: P
authors.lastname: De Los Rios
authors.forename: Pierre
authors.initials: P
authors.lastname: Goloubinoff
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D048888:Fireflies / Q000235:genetics / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D008156:Luciferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D017510:Protein Folding; D055550:Protein Stability
chemical_list: C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008156:Luciferases; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli