Biochemical Characterization of Symmetric GroEL-GroES Complexes: EVIDENCE FOR A ROLE IN PROTEIN FOLDING

Oscar Llorca,José L Carrascosa,José M Valpuesta

doi:10.1074/jbc.271.1.68

Abstract

When chaperonins GroEL and GroES are incubated under functional conditions in the presence of ATP (5 mM) and K+ (150 mM), GroEL-GroES complexes appear in the incubation mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or symmetric (1:2 GroEL:GroES oligomer ratio). The percentage of symmetric complexes present is directly related to the [ATP]/[ADP] ratio and to the K+ concentration. Kinetic analysis shows that there is a cycle of formation and disappearance of symmetric complexes. A correlation between the presence of symmetric complexes in the incubation mixture and its rhodanese folding activity suggests some active role of these complexes in the protein folding process. Accordingly, under functional conditions, symmetric complexes are found to contain denatured rhodanese. These data suggest that binding of substrate inside the GroEL cavity takes place before the symmetric complex is formed.

Highlights

When chaperonins GroEL and GroES are incubated under functional conditions in the presence of ATP (5 mM) and K؉ (150 mM), GroEL-GroES complexes appear in the incubation mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or symmetric (1:2 GroEL: GroES oligomer ratio)
Folding experiments show that both symmetric and asymmetric complexes are always present in the incubation mixture under functional conditions and that, regardless of the percentage of each type of complex, initial folding rates are similar, suggesting that both complexes could be implicated in the folding cycle
It has been previously shown that both asymmetric and symmetric complexes appear under folding conditions, whereas only asymmetric complexes are found in the presence of ADP or ATP␥S1 (2 mM, final concentration) (Llorca et al, 1994)

Summary

EXPERIMENTAL PROCEDURES

Purification of GroEL and GroES—E. coli chaperonins were obtained from a pOF39 plasmid harboring E. coli strain that overexpresses both. Sample Preparation and Electron Microscopy of GroEL-GroES Complexes—GroEL (0.3 ␮M final concentration) and GroES (1:2 molar ratio) were incubated in 50 mM Tris-HCl, pH 7.5, with different concentrations of MgCl2, KCl, and ATP as indicated in each experiment. For experiments with labeled rhodanese, complexes were obtained after cross-linking as indicated below. Cross-linking of GroEL and GroEL-GroES Complexes with Glutaraldehyde—Oligomers of GroEL (0.2 ␮M final concentration) either alone or with 125I-labeled rhodanese (equimolar to GroEL) were incubated in 50 mM Tris-HCl, pH 7.5, in the presence of 0.08% (w/v) glutaraldehyde (Sigma) for 20 min at 37 °C. After adding denatured 125I-labeled rhodanese (equimolar to GroEL), the mixture was incubated for 1 min at room temperature and crosslinked as indicated above. GroEL (0.3 mM final concentration) and GroES (1:2 molar ratio) were incubated in the presence of Mg2ϩ, Kϩ, and ATP for 15 min. The total number of particles for each experiment was 200

RESULTS

The abbreviations used are

DISCUSSION