The 4,4'-dipyridyl disulfide-induced formation of GroEL monomers is cooperative and leads to increased hydrophobic exposure.

The molecular chaperone, GroEL, is completely disassembled into monomers by the addition of 4,4'-dipyridyl disulfide. The dissociation leads to monomers in a kinetically controlled process. The additions of functional ligands of GroEL such as Mg(2+) or adenine nucleotides produced differences in the observed rates, but at the end of the kinetics, the dissociation was complete. In addition to the information obtained from native gels, the fluorescent probe bis-ANS was utilized to follow the monomer formation. The results demonstrate that the formation of monomers was associated with the exposure of hydrophobic surfaces. This assessment was possible without the use of added chaotropes, such as urea, to dissociate GroEL. Dissociation kinetics were also followed by light scattering. The kinetics of dissociation of the 14mer are cooperative with respect to the concentration of 4,4'-DPDS. Thermodynamic parameters for the kinetic process gave a free energy of activation (DeltaG) of 19.3 +/- 1.2 kcal mol(-1), which was decomposed to an enthalpy of activation (DeltaH) of 19.30 +/- 1.2 kcal mol(-1) and an entropy of activation (DeltaS) of -8.2 +/- 3.9 cal mol(-1) K(-1). We conclude that the dissociation of GroEL observed in this investigation is an enthalpy-controlled process.

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL.

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.