HYDROPHOBIC SURFACES THAT ARE HIDDEN IN CHAPERONIN CPN60 CAN BE EXPOSED BY FORMATION OF ASSEMBLY-COMPETENT MONOMERS OR BY IONIC PERTURBATION OF THE OLIGOMER

The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and bin ds tightly one to two molecules of the fluorescent hydrophobic reporte r, 1,1'-bi(4-anilino)naphthalene- 5,5'-disulfonic acid (bisANS). The 1 4-mer is resistant to proteolysis by chymotrypsin, and none of the thr ee sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. Wh en monomers of Cpn60 that are folded and competent to participate in p rotein folding are formed by low concentrations of urea (< 2.5 M), the hydrophobic exposure increases to accommodate approximately 14 molecu les of bisANS/14-mer, the binding affinity for bisANS decreases, and 1 sulfhydryl group/monomer reacts with 6-iodoacetamidofluorescein. Thes e monomers display limited proteolysis by chymotrypsin at several poin ts within a hydrophobic sequence centered around residue 250 to produc e a relatively stable N-terminal fragment (congruent to 26 kDa) and a partially overlapping C-terminal fragment (congruent to 44 kDa). The e xposure of hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic exposure more effectively than urea without dissociation of Cpn(60). For example, subdenaturing concentrations of guanidinium c hloride (less than or equal to 0.75 M) or the stabilizing salt, guanid inium sulfate, as well as NaCl or KCl are effective. The trivalent cat ion, spermidine, induces maximum exposure at 5 mM. The results suggest that hydrophobic surfaces can be involved in stabilizing the oligomer and/or in binding proteins to be folded, and they are consistent with suggestions that amphiphilic structures, presenting hydrophobic surfa ces within a charged context, would be particularly effective in bindi ng to Cpn60.