Nucleotide-dependent complex formation between the Escherichia coli chaperonins GroEL and GroES studied under equilibrium conditions.
Binding of heptameric GroES to the tetradecameric chaperonin GroEL in the absence or presence of nucleotides was investigated by analytical ultracentrifugation. In the absence of nucleotides, the association constant for the binding of GroES to GroEL, K1, was found to be approximately equal to 3 x 10(5) M(-1). The binding of a second GroES heptamer with only one-fourth the affinity of the first one can be neglected at subequimolecular concentrations relative to GroEL. Under these conditions, mainly an asymmetric "bullet"-shaped complex is formed [see also Schmidt et al. (1994) Science 265, 656-659]. In the presence of ADP or ATP analogues such as ATP-gamma-S or AMP-PNP, the affinity to bind GroES increases by at least 2 orders of magnitude depending on the nucleotide concentration. With increasing GroES:GroEL ratios in the presence of 1 mM ATP analogue, up to two GroES oligomers were bound to one GroEL oligomer, forming the symmetrical "American football"-shaped complex with apparently high affinity for the first GroES ring and considerably lower for the second one. These are the first results that provide an accurate and quantitative description of the equilibrium between asymmetrical and symmetrical complexes at relatively high concentrations of GroEL and GroES that are proposed to exist in vivo. We suggest that the increased affinity of GroEL for GroES plays a role in releasing substrate proteins from the central cavity of GroEL after folding under "non-permissive" conditions.
Behlke
J
J
Institute of Biology, Humboldt-University, Berlin, FRG. behlke@mdc-berlin.de
Ristau
O
O
Schönfeld
H J
HJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008970:Molecular Weight
D018835:Chaperonin 10; D018834:Chaperonin 60; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate