EF-Hand Mimicking Calcium Binding Polymer

There are four EF-hand polypeptides in calmodulin, a natural ubiquitous calcium binding protein that activates the enzymes involved in Ca²⁺-mediated signal transduction. An EF-hand polypeptide has six carboxylate functional groups in the middle loop region between two rigid polypeptides. In this study, a calcium binding polymer (CBP) with a structure of poly(L-alanine)-poly(L-alanine-co-L-glutamic acid)-poly(ethylene glycol)-poly(L-alanine-co-L-glutamic acid)-poly(L-alanine) (PA-PAE-PEG-PAE-PA; A_11.1-A_3.4E_3.2-EG_40.1-A_3.4E_3.2-A_11.1) was synthesized by mimicking the EF-hand polypeptide. The 6-7 carboxylate functional groups from PAE are expected to form a binding site for Ca²⁺. As the Ca²⁺ bound to CBP, small changes in the circular dichroism spectra and ¹³C NMR spectra were observed, indicating that Ca²⁺ binding to CBP induced changes in the conformation of CBP. The binding constant of CBP to Ca²⁺ was investigated by using the competitive binding of 2,2′, 2″, 2⌄ -{ethane-1,2-diylbis[oxy(4-bromo-2,1-phenylene)nitrilo]} tetraacetic acid (5,5-Br₂-BAPTA). The binding constant obtained with a CaLigator program by least-squares fitting of the absorbance profile as a function of Ca²⁺ concentration was 5.1 × 10⁵ M^-1, which was similar to that of calmodulin. The selectivity of CBP for metal ion binding was compared among Ca²⁺, Cu²⁺, and Zn²⁺. The binding constant was obtained through a similar competitive binding study with murexide. The binding constants for Ca²⁺, Cu²⁺, and Zn²⁺ were 7.0 × 10⁵, 4.2 × 10⁵, and 1.7 × 10⁵ M^-1, respectively, indicating 2-4-fold higher selectivity of CBP for Ca²⁺ compared to Cu²⁺ and Zn²⁺. The CBP has selectivity for Ca²⁺, and binding affinity for Ca²⁺ was similar to the biological Ca²⁺ binding motif of calmodulin. (Figure Presented).