Kinetics of peptide binding to the bovine 70 kDa heat shock cognate protein, a molecular chaperone.
We have measured the kinetics of binding and release of a fluorescently labeled seven-residue peptide (fluorescein-FYQLALT) to recombinant bovine heat shock cognate protein (Hsc70); additionally, we have determined the effect of peptide binding on the kinetic rate constants of individual steps of the Hsc70 ATPase cycle. In the presence of MgADP, peptide binding is a two-step process; the first step results in a low-affinity peptide-Hsc70 complex (Kd calcd approximately 14 microM), while the second step locks the peptide into a higher-affinity complex (Kd = 4.3 microM). In the presence of MgATP, peptide binding is a one-step process which yields a peptide-Hsc70 complex with an affinity of approximately 40-50 microM. The bimolecular rates of initial peptide-Hsc70 association differ less than 2-fold in the presence of MgADP and MgATP. Peptide binding increases the rates of ATP hydrolysis and product release in the Hsc70 ATPase cycle. Taken together with earlier results, these data suggest a model for the interaction of Hsc70 with peptides in which (i) with MgATP there is significant interaction between the carboxy terminal peptide binding domain and the amino terminal ATPase domain of Hsc70 such that the effect of peptide binding is transmitted to the ATPase domain (resulting in increased rates of ATP hydrolysis and product release) and, reciprocally, the ATPase domain constrains the peptide binding domain to a low-peptide affinity conformation; and (ii) with MgADP, the peptide binding domain is less constrained by the ATPase domain, allowing capture of peptides in complexes with significantly slower "off " rates than in the presence of MgATP.
Takeda
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S
Department of Structural Biology, Stanford University School of Medicine, California 94305-5400, USA.
McKay
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DB
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D005452:Fluoresceins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D005452:Fluoresceins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate