title: Spectroscopic and thermodynamic measurements of nucleotide-induced changes in the human 70-kDa heat shock cognate protein.
abstract: Hsp70 alternates between an ATP-bound state in which the affinity for substrate is low and an ADP-bound state in which the affinity for substrate is high, as a result Hsp70 assists the protein folding process through nucleotide-controlled cycles of substrate binding and release. In this work, we describe the cloning and purification of the human 70-kDa heat shock cognate protein, Hsc70, and the use of circular dichroism, intrinsic emission fluorescence, and isothermal titration calorimetry to characterize conformational changes induced by ADP and ATP binding. Binding of either ADP or ATP were not accompanied by a net change in secondary structure suggesting that the conformational rearrangement caused by nucleotide binding is localized. MgADP or MgATP had a greater effect in the stability at stress temperatures than ADP or ATP did. Isothermal titration calorimetry data pointed out that Hsc70 had a lower affinity for ATP (KD=710 nM) than for ADP (KD=260 nM).
authors.affiliation: Laboratório Nacional de Luz Síncrotron, P.O. Box 6192, Zip code 13084-971, Campinas SP, Brazil.
authors.forename: Júlio C
authors.initials: JC
authors.lastname: Borges
authors.forename: Carlos H I
authors.initials: CH
authors.lastname: Ramos
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D017186:Titrimetry / Q000379:methods
chemical_list: D050883:HSC70 Heat-Shock Proteins; D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate