THERMODYNAMICS OF METAL-ION BINDING AND DENATURATION OF A CALCIUM-BINDING PROTEIN FROM ENTAMOEBA-HISTOLYTICA

The thermodynamics of tie binding of calcium and magnesium ions to a c alcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) a t 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca 2+, both of which are exothermic in nature and with positive cooperati ve interaction between them. Two other high affinity sites for Ca2+ ex ist of which one is endothermic and the other exothermic, again with p ositive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where C aBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2 + and a Ca2+ titration employing intrinsic tyrosine fluorescence of th e protein, The enthalpy of titration of magnesium in the absence of ca lcium is single site and endothermic in nature. In the case of the tit rations performed using protein presaturated with magnesium, the amoun t of heat produced is altered. Further, the interaction between the hi gh-affinity sites changes to negative cooperativity. No exchange of he at was observed throughout the addition of magnesium in the presence o f 1 mM calcium, Titrations performed on a cleaved peptide comprising t he N-terminus and the central linker show the existence of two Ca2+ sp ecific sites, These results indicate that this CaBP has one high-affin ity Ca-Mg site, one high-affinity Ca-specific site, and two low-affini ty Ca-specific sites. The thermodynamic parameters of the binding of t hese metal ions were used to elucidate the energetics at the individua l site(s) and the interactions involved therein at various concentrati ons of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6 .5 M. Unfolding of the protein was also monitored by titration calorim etry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to C a2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this pr otein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control exper iments with change in ionic strength by addition of KCI in the range 0 .25-1 M show the existence of four sites with altered ion binding para meters.