Protein substrate binding induces conformational changes in the chaperoninGroEL - A suggested mechanism for unfoldase activity

Chaperonins are molecules that assist proteins during folding and protect t hem from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase LI (HCA II), which ind uces unfolding of the enzyme. We focused on conformational changes that occ ur in GroEL during formation of the GroEL-HCA II complex. We measured the r ate of GroEL cysteine reactivity toward iodo[2-C-14]acetic acid and found t hat the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxyl-2,2,5,5-tetramethyl -3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurre d because buried cysteine residues become accessible during HCA LI binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhib ited decreased fluorescence anisotropy upon HCA II:binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modifi ed GroEL with the spin label N-(1-oxyl-2,2,5,5 -tetramethyl-3-pyrrolidinyl) iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amin o)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these r esults show that conformational changes occur in the chaperonin as a conseq uence of protein substrate binding. Together with previous results on the u nfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of th e protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II .