Mutation Ala2 → Ser Destabilizes Intersubunit Interactions in the Molecular Chaperone GroEL

In vitro dissociation and self‐assembly of three chaperonin 60s: the role of ATP

“…It is no surprise, therefore, that the binding of Mg-adenine nucleotides and/or the cycles of ATP hydrolysis can influence the structure of the 14-mers. Reversible ATP hydrolysis-driven transitions between two conformations of the 14-mers have recently been discovered which are believed to be directly connected to the functioning of GroEL [15,27,30,[32][33][34][35]. Of the two states the 'tense' one (T) is more stable, while the 'relaxed' (R) one possesses higher affinity to the nucleotides and the substrates non-folded proteins [27].…”

“…Reversible ATP hydrolysis-driven transitions between two conformations of the 14-mers have recently been discovered which are believed to be directly connected to the functioning of GroEL [15,27,30,[32][33][34][35]. Of the two states the 'tense' one (T) is more stable, while the 'relaxed' (R) one possesses higher affinity to the nucleotides and the substrates non-folded proteins [27]. Since the oligomers of all three proteins tested in this study are destabilized to some extent in the presence of Mg-ATP or Mg-ADP, it is suggested that the nucleotide-dependent transitions between T-and R-like states (from which the latter can more easily dissociate into free monomers) represent a common feature of all homologous chaperonins.…”

“…Size-exclusion chromatography was used in preference to native PAGE because the results can easily be quantitated, and no increase in the protein concentration (which potentially can cause artefacts) occurs at the start of the separation [17]. Native (non-denaturing) PAGE on a 7.5% gel performed as described in [27] was used whenever the chromatographic approach was not applicable.…”

“…Each micro cell was then subjected to dialysis (2 h at 23°C) against 1 ml of buffer A containing the additives as indicated. Glycerol (20% v/v) was added to the buffer (except where otherwise indicated) to minimise the osmotic dilution of the samples• At the end of the dialysis EDTA (up to 15 mM) was added to stop the effect of Mg-adenine nucleotides [27], and the samples were analysed by non-denaturing PAGE as described above.…”

“…On the other hand, a massive dissociation of the cpn60 oligomers caused by Mg-ATP is observed in the stromal extracts of pea chloroplasts [12,19,20]. Recently it has been reported that Mgadenine nucleotides (ATP or ADP) enhance the dissociation of GroEL 14-mers in the presence of urea [27]. A similar approach was employed in this study to quantitate relative effects of different adenine nucleotides on the three chaperonin proteins.…”