The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES

Abstract

In order to understand the role of ATP hydrolysis of the chaperonin GroEL during protein folding, we have studied GroEL-GroES complex formation in the presence of ATP or ADP by using capillary electrophoresis and surface plasmon resonance. Capillary electrophoresis analysis showed that the GroEL 14-mer and GroES 7-met formed a 1:1 complex in the presence of ATP. In the presence of ADP, both the association and dissociation rates of the complex were slower by about one order of magnitude than the rates in the presence of ATP at 25 ° C. The implications of such a stable complex on the overall mechanism of chaperonin function are discussed.

Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60

“…However, it can be applied to determine the affinity between cpn60 and cpn10 in the presence of ADP, where the chaperonin complex has a half-life of Ϸ5 h (12,38). We find that the K d for the E. coli GroEL͞GroES complex determined by sucrose density centrifugation agrees well with published data (12,39,40). The yeast hsp60͞hsp10 complex is of similar stability.…”

The affinity of the GroEL/GroES complex for peptides under conditions of protein folding

“…The affinity of the GroEL/ADP complex for the peptides (static situation) was found to be enhanced relative to the dynamic situation under conditions of ATP hydrolysis. This finding is consistent with the fact that GroEL displays weaker binding for ADP relative to ATP [32,33], and that higher concentrations of ADP are required for the transition of high to low affinity complexes [12].…”

“…GroEL and GroES are thought not to associate very tightly in the absence of nucleotides [12,32,36,37] (buffer A), although these complexes may form more readily in buffer B for reasons of salt shielding of electrostatic repulsion between the two acidic proteins [22]. Given the fact that the peptide binding sites of GroEL are well known to double up as GroES binding sites [38–41], GroEL/GroES complexes, once formed, are likely to offer only half of the binding regions [17].…”

Affinity Capillary Electrophoresis: A Sensitive Tool for the Study of Molecular Interactions and Its Use in Microscale Analyses

“…When the interacting molecules have large differtrimethylammoniumpropyl sulfonate (56). Inclusion of an ionic surfactant in the separation buffer, a routine ences in their mobility, it may be difficult to detect all of the free and complexed species in the same electrotechnique for the separation of low-molecular-weight substances in micellar electrokinetic chromatography phoretic run.…”

“…For a system with slow on-and-off kinet-by the disappearance of the peak of either of the free ics, such as most high-affinity systems, the equilib-components when it is mixed with the other, and by rium-mixture analysis is preferable and will be dis-its emergence after the saturation of the binding site cussed in this section. For a system having relatively of the other (56,(60)(61)(62). rapid on-and-off kinetics, a typical feature of low-affinLabeling of one component with a fluorophore enity complexes, the mobility-change analysis should be ables the selective detection of the complex even when selected and will be discussed in the next section.…”