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. Metalfree 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) resultsshow 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.

Concanavalin A (ConA) is a plant lectin having industrial and biological applications. Concanavalin A changes conformation upon exposure to different stress conditions, like exposure to sodium dodecyl sulphate, guanidine hydrochloride, varying hydronium ion potential, etc. The conformational changes were studied using circular dichroism spectroscopy and the structural rigidity of ConA was explored using fluorescence spectroscopy, taking tryptophan as an intrinsic and 8-anilino-1-naphthalenesulfonic acid as an extrinsic fluorescence probes, in different stress conditions. ConA loses the quaternary structure in all the studied stress conditions, which further leads to denaturation of the protein. However, the mechanism of denaturation varied with the studied conditions, like different SDS concentrations and hydronium ion potentials, wherein the proteinundergoes a conformational rearrangement from β-sheet to α-helix. Moreover, GdnHCl triggered complete denaturation of ConA into a predominantly random coil conformation. The results suggested that denaturation of ConA follows different pathways depending on the chemical properties and concentrations of the denaturants used.