title: Kinetics of nucleotide-induced changes in the tryptophan fluorescence of the molecular chaperone Hsc70 and its subfragments suggest the ATP-induced conformational change follows initial ATP binding.
abstract: The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single-cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment.
authors.affiliation: Beckman Laboratories for Structural Biology, Department of Structural Biology, Stanford University School of Medicine, California 94305, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Ha
authors.forename: D B
authors.initials: DB
authors.lastname: McKay
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D005453:Fluorescence; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014364:Tryptophan; D000255:Adenosine Triphosphate