title: Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica.
abstract: Sequence analysis and metal ion binding studies reported earlier have established that the calcium binding protein (CaBP) from the parasitic ameboid Entamoeba histolytica protein has four canonical EF hand motifs which bind calcium. Equilibrium denaturation studies on both the apo and the holo forms of this protein indicate the presence of stable transition intermediates at low denaturant concentrations as revealed by the binding of the non-specific hydrophobic dye ANS. Fast reaction kinetics shows that the binding of the Gdn+ ions at or near the Ca2+ sites in the N-terminal domain influences metal ion binding to the sites in the C-terminal domain. Isothermal calorimetric titrations performed using low GdnHCl concentrations reveal the presence of two binding sites of low affinity, both being endothermic in nature. Thus the stabilization of CaBP observed at low GdnHCl concentration represents a native-like intermediate, with the Gdn+ ions mimicking Ca2+ binding at the N-terminal domain of this protein.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
authors.forename: B
authors.initials: B
authors.lastname: Gopal
authors.forename: J V
authors.initials: JV
authors.lastname: Krishna Rao
authors.forename: C J
authors.initials: CJ
authors.lastname: Thomas
authors.forename: A
authors.initials: A
authors.lastname: Bhattacharya
authors.forename: S
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: M R
authors.initials: MR
authors.lastname: Murthy
authors.forename: A
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002151:Calorimetry; D004748:Entamoeba histolytica / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D008274:Magnesium; D019791:Guanidine; D002118:Calcium