Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica

Sequence analysis and metal ion binding studies reported earlier have estab lished that the calcium binding protein (CaBP) from the parasitic ameboid E ntamoeba histolytica protein has four canonical EF hand motifs which bind c alcium. Equilibrium denaturation studies on both the apo and the hole forms of this protein indicate the presence of stable transition intermediates a t low denaturant concentrations as revealed by the binding of the non-speci fic hydrophobic dye ANS. Fast reaction kinetics shows that the binding of t he Gdn(+) ions at or near the Ca2+ sites in the N-terminal domain influence s metal ion binding to the sites in the C-terminal domain. Isothermal calor imetric titrations performed using low GdnHCl concentrations reveal the pre sence of two binding sites of low affinity, both being endothermic in natur e. Thus the stabilization of CaBP observed at low GdnHCl concentration repr esents a native-like intermediate, with the Gdn(+) ions mimicking Ca2+ bind ing at the N-terminal domain of this protein. (C) 1998 Federation of Europe an Biochemical Societies.