Ca 2+ and Mg 2+ binding induce conformational stability of Calfumirin-1 from Dictyostelium discoideum

The apo-Calfumirin-1 (CAF-1) binds to Ca2+ with high affinity and also to Mg2+ with high positive cooperativity. The thermal unfolding curves of wtCAF-1 monitored at neutral pH by CD spectroscopy are reversible and show different thermal stabilities in the absence or presence of Ca2+ and Mg2+ ions. Metal-free wtCAF-1 shows greater thermal stability than EF-IV mutant protein. We observed that GdnHCl-induced unfolding of apo-wtCAF-1 monitored by CD and fluorescence spectroscopies increases co-operative folding with approximately same C-m values. Binding of Ca2+ and Mg2+ ions to CAF-1 dramatically altered the fluorescence and CD spectra, indicating metal ion-induced conformational changes both in the wild-type and mutant proteins. The hydrophobic probe, ANS is used to observe alteration in surface hydrophobicity of the protein in different ligation states. In apo-wtCAF-1, the exposed hydrophobic surfaces are able to bind ANS which is in contrast to the unfolded or the metal ions ligated conformations. Isothermal titration calorimetry (ITC) results show two possible independent binding sites of comparable affinity for the metal ions. However, their binding to the EF-IV E helix-loop-F helix mutant apo-protein happens with different affinities. The present study demonstrates that Ca2+ or Mg2+ binding plays a possible role in the conformational stability of the protein.