Elucidation of calcium coordination mechanisms in cod bone-derived peptides and site-directed mutants using quantum chemistry.

The objective of this study was to elucidate the Ca-binding mechanisms of cod bone-derived peptides and their site-directed mutants using experimental and quantum-chemical approaches. Isothermal titration calorimetry showed Ca-binding was exothermic, spontaneous at 25 °C, driven mainly by ionic interactions. Binding constants for DYGGEY and GESGEAGDR were 1.120 × 103 and 1.024 × 103 M-1, with stoichiometries of 2 Ca2+ per peptide. Mutants DYGEGY, AYGGAY, and GASGAAGAR showed Ca-binding capacities of 4.64 ± 0.42, 0.73 ± 0.37, and 1.07 ± 0.55 μg/mg, underscoring the importance of carboxyl groups. DFT calculations identified carboxyl oxygens as binding sites, stabilized by electrostatic and van der Waals forces. Multidentate coordination involving hydroxyl oxygens appeared in DYGGEY, GESGEAGDR, and DYGEGY, while amide oxygen coordination led to weaker interactions in AYGGAY and GASGAAGAR. Electronic structure analysis confirmed carboxyl regions as reactive sites, offering insights for designing Ca-binding peptides for nutritional applications.