Chaperonin-affected refolding of alpha-lactalbumin: Effects of nucleotidesand the Co-chaperonin GroES

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperon in GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. T he refolding reactions induced by stopped-flow pH jumps were monitored by a lpha-lactalbumin tryptophan fluorescence. The simple single-exponential cha racter of the free-refolding kinetics of the protein allowed us to quantita tively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for bind ing between GroEL and the refolding intermediate of alpha-lactalbumin by th e non-linear least-squares method. When GroES was absent, the interaction b etween GroEL and alpha-lactalbumin could be well represented by a "cooperat ive-binding" model in which GroEL has two binding sites for alpha-lactalbum in with the affinity of the second site being tenfold weaker than that of t he first, so that there is negative cooperativity between the two sites. Th e affinity between GroEL and a-lactalbumin was significantly reduced when A TP was present, while ADP and AMP-PNP did not alter the affinity. A compari son of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to th e nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between Gro EL and the refolding intermediate of alpha-lactalbumin. The affinity at a s aturating concentration of ADP or AMP-PNP was about ten times lower than wi th GroEL alone. The ADP concentration at which the acceleration of the GroE L/ES-affected refolding of alpha LA was observed, was higher than the conce ntration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself i s not enough to reduce the affinity for alpha-lactalbummin, and that furthe r binding of the nucleotide to the GroEL/ES complex is required to reduce t he affinity. (C) 1999 Academic Press.