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. Proteins 2011; © 2011 Wiley‐Liss, Inc.