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 e pical domain fragment of GroEL, or minichaperone (residues 191-345), was in vestigated and compared with that of the GroEL tetradecamer. Although a tot al 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 lo w bis-ANS concentrations, it is evident that the native apical domain is co nverted to an intermediate conformation with increased hydrophobic surfaces . This intermediate binds additional bis-ANS molecules. Tyrosine fluorescen ce detected denaturation demonstrated that bis-ANS can destabilize the apic al domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosin e 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 t hat 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 d ue to quaternary structure.