Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate

The denaturation of the heat shock protein DnaK induced by guanidine hydrochloride (Gdn-HCl) was investigated by circular dichroism, fluorescence, size-exclusion HPLC, and dynamic light scattering. DnaK unfolding takes place in two discrete steps. The midpoint (Cm) of the first transition (0.5 M) was shifted to higher denaturant concentrations (0.8 M) in the presence of Mg/ADP or Mg/ATP, whereas the second transition (Cm = 1.6 M) was unaffected by nucleotides. An intermediate state which continuously expands with increasing Gdn.HCl concentration was observed; its relation to molten globules is discussed. In addition, a direct correlation between molecular volume and ellipticity at 222 nm was found, regardless of the conformational state (native, intermediate, unfolded); the implications of these findings are discussed. The unfolding of DnaK is best explained by a hierarchical model of unfolding.