Fig. 1: Binding affinity studies of hepta-peptide with Ca²⁺.

a, b Isothermal titration calorimetric data when various equivalents of CaCl₂ (20 mM in H₂O) were added to a DDDEEKC aqueous solution (1.0 mM) at 20 °C. The red line in b represents a nonlinear fitting curve based on a sequential binding site model (N = 5). Partial carbon nuclear magnetic resonance (¹³C NMR) spectra of DDDEEKC (c) and the mixture of DDDEEKC and CaCl₂ at molar ratios of 1:1 (d), 1:3 (e), and 1:5 (f) in d₆-DMSO at 20 °C; chemical shift changes of carboxylic acids and amides of the hepta-peptide are indicated by black dashed arrows. The NMR attribution of each group is determined by quantum chemistry calculation [Gassiness, density functional theory (DFT), at 6–31 g level of theory]