The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP.

Abstract

The yields of active dodecameric glutamine synthetase (GS) are significantly increased when in vitro folding is initiated in the presence of the Escherichia coli groE chaperonins and ATP (37 degrees C). To observe the effects of chaperonins and ATP on GS assembly, the GS assembly intermediates were separated by nondenaturing gel electrophoresis, visualized by Western analysis, and studied as a function of time. The form of GS that was initially released from groEL is monomeric. After the monomers formed dimers, active GS oligomers were assembled by the association of assembly competent dimers with higher order even-numbered oligomers until the dodecamer was formed. When ATP was added to the groEL.GS complex (no groES), a groEL.GS complex remained visible for up to 30 min after the renaturation was initiated. This slow disappearance of the groEL.GS complex is consistent with observed lags in both the GS activity regain profile and the assembly-dependent increase in GS tryptophan fluorescence. When groES was present, the addition of ATP resulted in the disappearance of observable complex at early sample times (< 2 min). Concomitantly, the rates of the regain of GS activity and the GS-dependent increase in tryptophan fluorescence intensity showed substantial accelerations. These results indicate that groES facilitates GS assembly from groEL by inducing the rapid release of GS from groEL, which in turn increases the concentration of assembly competent GS monomers. In addition, groES can initiate renaturation of GS from the groEL.GS arrested complex in the presence of ADP. When chaperonin-dependent GS renaturation was initiated with ATP or ADP (> or = 2 mM), the rates were identical. Since ATP hydrolysis is not absolutely required, the combined binding energies of groES and ATP (or ADP) appear to be sufficient to weaken the binding affinity of groEL for GS subunits and facilitate the release and refolding of assembly competent GS monomers from groEL.