A conundrum has arisen in the study of the structural states of the GroEL-GroES chaperonin machine: When either ATP or ADP is added along with GroES to GroEL, the same asymmetric complex, with one ring in a GroES-domed state, is observed by either x-ray crystallographic study or cryoelectron microscopy. Yet only ATP͞ GroES can trigger productive folding inside the GroES-encapsulated cis cavity by ejecting bound polypeptide from hydrophobic apical binding sites during attendant rigid body elevation and twisting of these domains. Here, we show that this difference occurs because polypeptide substrate in fact presents a load on the apical domains, and, although ATP can counter this load effectively, ADP cannot. We monitored apical domain movement in real time by fluorescence resonance energy transfer (FRET) between a fixed equatorial fluorophore and one attached to the mobile apical domain. In the absence of bound polypeptide, addition of either ATP͞GroES or ADP͞GroES to GroEL produced the same rapid rate and extent of decrease of FRET (t 1/2 < 1 sec), reflecting similarly rapid apical movement to the same end-state and explaining the results of the structural studies, which were all carried out in the absence of substrate polypeptide. But in the presence of bound malate dehydrogenase or rhodanese, whereas similar rapid and extensive FRET changes were observed with ATP͞GroES, the rate of FRET change with ADP͞GroES was slowed by >100-fold and the extent of change was reduced, indicating that the apical domains opened in a slow and partial fashion. These results indicate that the free energy of ␥-phosphate binding, measured earlier as 43 kcal per mol (1 cal ‫؍‬ 4.184 J) of rings, is required for driving the forceful excursion or ''power stroke'' of the apical domains needed to trigger release of the polypeptide load into the central cavity.

The current mechanistic model of chaperonin-assisted protein folding assumes that the substrate protein in the cage, formed by GroEL central cavity capped with GroES, is isolated from outside and exists as a free polypeptide. However, using ATPase-deficient GroEL mutants that keep GroES bound, we found that, in the rate-limiting intermediate of a chaperonin reaction, the unfolded polypeptide in the cage partly protrudes through a narrow space near the GroEL/GroES interface. Then, the entire polypeptide is released either into the cage or to the outside medium. The former adopts a native structure very rapidly and the latter undergoes spontaneous folding. Partition of the in-cage folding and the escape varies among substrate proteins and is affected by hydrophobic interaction between the polypeptide and GroEL cavity wall. The ATPase-active GroEL with decreased in-cage folding produced less of a native model substrate protein in Escherichia coli cells. Thus, the polypeptide in the critical GroEL-GroES complex is neither free nor completely confined in the cage, but it is interacting with GroEL's apical region, partly protruding to outside.

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeFx) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeFxstops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP·BeFxand an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADPplusBeFx, however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP·BeFxand an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATPplusBeFx. Thus, BeFxstabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP·Pinucleotide states in the functional cycle of GroEL.

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