In the present work we describe a simple experimental model protein tailored to study the formation of metal ion-assisted π−π interaction during ligand-induced protein folding. The model system is based on a calcium-binding protein, SPM, a self-processing module, derived from the internal segment of the bacterial FrpC protein, which mediates a Ca2+-dependent autocatalytic cleavage of the highly specific Asp-Pro peptide bond and covalent linkage of the carboxy-terminal group of the splinter segment to ε-amino group of a lysine residue of an adjacent protein. SPM is a polypeptide that is intrinsically disordered in the absence of calcium and folds upon binding of Ca2+ ions into a compact and stable structure. The cleavage of the Asp-Pro bond is accompanied with the Ca2+-dependent folding of SPM that is unambiguously characterized by the formation of Ca2+-assisted π−π interaction between a pair of unique tryptophan residues. Therefore, Ca2+-dependent folding linked to enzymatic activity would prove SPM to be an excellent model to investigate the formation of Ca2+−π−π interaction during the ligand-induced transition from an unfolded to the folded conformation.