title: Multiple molecules of Hsc70 and a dimer of DjA1 independently bind to an unfolded protein.
abstract: Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound (14)C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.
authors.affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan. terada@gpo.kumamoto-u.ac.jp
authors.forename: Kazutoyo
authors.initials: K
authors.lastname: Terada
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Oike
mesh_terms: D000818:Animals; D002482:Cellulose / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry*; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D020349:Surface Plasmon Resonance
chemical_list: D003432:Cross-Linking Reagents; C107481:DNAJA1 protein, human; C494639:Dnaja1 protein, mouse; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018832:Molecular Chaperones; D002482:Cellulose