Engineering naturally occurring proteins enables us to customize affinity domains according to our specific needs, tailoring them to become an important tool in a wide array of applications limited merely by our creativity. Nature has an elegant way to control protein activity by creating a functional change through alteration of a protein’s tertiary structure, a change which could be induced upon binding to a target molecule or through the interaction with ions that help stabilize the protein’s structure. Inspired by nature where many protein-protein interactions have evolved to be dependent on metal ions we made an engineering effort to introduce a calcium-binding loop into a small, 56-amino acid, domain of Streptococcal Protein G. Through a combination of rational protein design and randomization, selection by E. coli display in combination with fluorescence-activated cell sorting, it was possible to isolate several domains where the inherent target affinity to antibodies and antibody fragments had been rendered calcium-dependent. Once depleted of calcium, the target interaction of these proteins is lost. Interestingly, structural determination of these novel domains using NMR revealed that the introduced calcium dependency was connected to the protein folding as well as the target binding. Designing binding moieties to confer calcium dependency is expanding and improving the era of combinatorial protein engineering, enabling development of proteins which target affinity could be tailored to be turned on or off based on application. This could prove to be highly valuable within many research fields such as in the creation of selective protein therapeutics or in the development of milder antibody purification procedures.