Deciphering calcium-binding behaviors of casein phosphopeptides by experimental approaches and molecular simulation.

Although specific groups in casein phosphopeptides (CPPs) have been identified to be essential for the calcium-binding capacity, how calcium binds with CPPs remains unclear. In the present study, experimental methods (UV-vis, FTIR and isothermal titration calorimetry) and molecular dynamic simulation were combined to reveal the calcium-binding behaviors of β-casein phosphopeptides (1-25) (P5) with the best capability in carrying calcium ions. We found that it could carry approximately six calcium ions, and the calcium-binding sites were primarily consisted of the carbonyl group of Glu-2 and the phosphate group of phosphorylated Ser-15, Ser-17, and Ser-19. An interesting finding was that calcium ions could be bound by three coordinated modes, including unidentate, bidentate and tridentate geometries, resulting in the strong binding abilities. The binding process of calcium ions to P5 was spontaneous with the binding free energies of -5.2 kcal/mol. Hydrophobic interaction was considered to be the major driving force for the calcium ion binding. The present study provides novel molecular insights into the current understanding and research of CPPs.