Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10.
The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.
Staniforth
R A
RA
Molecular Recognition Centre, University of Bristol, School of Medical Sciences, U.K.
Burston
S G
SG
Atkinson
T
T
Clarke
A R
AR
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018835:Chaperonin 10; D018834:Chaperonin 60; D001411:Geobacillus stearothermophilus; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000648:ultrastructure; D046911:Macromolecular Substances; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins; D013816:Thermodynamics
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D009711:Nucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase