GroE chaperonin-assisted folding and assembly of dodecameric glutamine synthetase.

The folding and assembly of Escherichia coli dodecameric glutamine synthetase is facilitated by the E. coli GroE chaperonins, GroEL and GroES. Since endogenous glutamine synthetase monomers are bound to GroEL immediately after cell lysis and are assembly competent, this strongly suggests that glutamine synthetase is an authentic substrate of the GroE chaperonins. At physiological temperatures, the in vitro reactivation of glutamine synthetase increases from 10 to 70-80% of the original activity when the chaperonin GroEL is included. Although nucleotide binding is sufficient to dissociate assembly competent glutamine synthetase monomers from GroEL, the addition of GroES substantially accelerates the dissociation, assembly, and reactivation. The interactions of glutamine synthetase monomers with the activated chaperonin are transient (t1/2 = 10 sec) and these monomers can be released from GroEL at high concentrations without misfolding or inappropriate aggregation. It has been found that the nucleotide-induced conformational change of GroEL is critical for folding success of glutamine synthetase because the simple displacement of glutamine synthetase monomers from the GroEL chaperonin with another protein substrate inhibits reactivation. During glutamine synthetase refolding, the "high affinity" nucleotide-free GroEL is most efficient in preventing initial folding intermediates from partitioning to off-pathway folding routes. Interestingly, the more physiologically relevant "low affinity" nucleotide-bound ((ATP/ADP) GroEL--GroES) complex is not as efficient at capturing the initial folding intermediates of glutamine synthetase. In contrast to glutamine synthetase, non-authentic "model" substrates such as mammalian mitochondrial rhodanese and mitochondrial malate dehydrogenase show no differences in folding efficiencies with either the "low affinity" or "high affinity" complexes. Besides the nature of the chaperonin complex itself, the mechanism of GroE-assisted folding is determined by the folding environment and, most importantly, by initial interactions of chaperonins with folding intermediates. Glutamine synthetase interacts only transiently with chaperonin complexes, while most of the "model" proteins exhibit relatively long interactions times. It may be indicative of a specific evolutionary selected mechanism of chaperonin-assisted folding (optimizing the folding kinetics), different from that observed with non-authentic chaperonin substrates. Since the kinetics of protein folding depends heavily on the solution environment, studies involving in vivo chaperonin substrates under conditions that closely mimic those found in the cell will be required to define and solve the physiologically relevant kinetic mechanism of chaperonin-assisted folding.