title: Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone.
abstract: The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.
authors.affiliation: Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan. tanaka@ip.kit.ac.jp
authors.forename: Naoki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Shota
authors.initials: S
authors.lastname: Nakao
authors.forename: Jean
authors.initials: J
authors.lastname: Chatellier
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Tani
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tada
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kunugi
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D010455:Peptides; C063842:dnaK protein, E coli; D019791:Guanidine