Abstract
The urea-induced dissociation and subsequent conformational transitions of the nucleotide-bound form of GroEL were studied by light scattering, 4,4'-bis(1-anilino-8- naphthalenesulfonic acid) binding, and intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM) stabilizes GroEL and leads to coordination of the structural transitions monitored by the different parameters. The midpoint of the light-scattering transition that monitored dissociation of the 14-mer with bound magnesium was raised to approximately 3 M, which is considerably higher than the ligand-free form of the protein, which exhibits a transition with a midpoint at approximately 2 M urea. Binding of ADP results in destabilization of the GroEL oligomeric structure, and complete dissociation of the 14-mer in the presence of 5 mM ADP occurs at about 2 M urea with the midpoint of the transition at approximately 1 M urea. The same destabilization by ADP and stabilization by Mg2+ were seen when the conformation was followed by the intrinsic fluorescence. Complexation with the nonhydrolyzable ATP analog, 5'-adenylimidodiphosphate gave an apparent stability of the quaternary structure that was between that observed with Mg2+ and that with ADP. The ADP-bound form of the protein demonstrated increased hydrophobic exposure at lower urea concentrations than the uncomplexed GroEL. In addition, the GroEL-ADP complex is more accessible for proteolytic digestion by chymotrypsin than the uncomplexed protein, consistent with a more open, flexible form of the protein. The implication of the conformational changes to the mechanism of the GroEL function is discussed.
Highlights
Molecular chaperones are proteins that can assist in the folding of other proteins (Hartl et al, 1994)
Two features of GroEL that have been suggested to be important for binding target proteins are 1) coordination of the multiple sites in the oligomer, and 2) conformational changes that could lead to exposure of interactive surfaces
Studies with fluorescent probes suggested that conformational changes in GroEL are required for extensive hydrophobic exposure, so that important functional aspects of chaperoninassisted refolding reside in the ease with which ligand interactions can trigger conformational changes (Horowitz et al, 1995)
Summary
Molecular chaperones are proteins that can assist in the folding of other proteins (Hartl et al, 1994). Release of a bound target protein is accompanied by GroEL-mediated ATP hydrolysis (Hartl et al, 1994), and some proteins require a second protein, the co-chaperonin GroES, which is a seven-subunit homooligomeric protein. Refolding of some other target proteins from their GroEL-polypeptide complexes can be induced just by addition of nucleotides. Proteins of this type include dihydrofolate reductase (Viitanen et al, 1991), tryptophanase (Mizobata et al, 1992), and the Fab fragment of a monoclonal antibody (Schmidt et al, 1992). Other investigators proposed the existence of two interconvertable forms of GroEL, one, stabilized by MgATP, which associates weakly with unfolded polypeptide, and a second destabilized by MgATP, which associates strongly with the target protein (Badcoe et al, 1991). Possible implications of these phenomena to the mechanism of GroEL function are discussed
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