Deciphering the calcium-binding mechanism of a novel cod bone-derived peptide: A synergistic experimental and computational analysis

A novel cod bone peptide GETGPA with high calcium chelating capacity (4.60 ± 1.06 μg/mg) was prepared and synthesized to elucidate the binding mechanism of peptide with Ca 2+ . The chelation sites, binding modes, and coordination bond formation mechanism were systematically investigated by experimental methods combined with molecular dynamics simulation and density functional theory calculation. FTIR and Raman spectra showed that the oxygen atoms of carboxyl groups were likely involved in the binding of GETGPA with Ca 2+ . Furthermore, mass spectrometry identified Glu-2 as the primary calcium binding site of GETGPA. NMR indicated that the free carboxyl group of Ala-6 and Glu-2 might be participated in Ca 2+ chelation. There were multiple chelating modes in the system where multiple GETGPA molecules coexisted with Ca 2+ . The coordination bond formed between GETGPA and Ca 2+ exhibited excellent chemical and thermodynamic stability. This study provided new insights into the binding mechanisms of peptides and calcium.