title: EF-Hand Mimicking Calcium Binding Polymer.
abstract: There are four EF-hand polypeptides in calmodulin, a natural ubiquitous calcium binding protein that activates the enzymes involved in Ca(2+)-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; A11.1-A3.4E3.2-EG40.1-A3.4E3.2-A11.1) was synthesized by mimicking the EF-hand polypeptide. The 6-7 carboxylate functional groups from PAE are expected to form a binding site for Ca(2+). As the Ca(2+) bound to CBP, small changes in the circular dichroism spectra and (13)C NMR spectra were observed, indicating that Ca(2+) binding to CBP induced changes in the conformation of CBP. The binding constant of CBP to Ca(2+) 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 Ca(2+) concentration was 5.1 × 10(5) M(-1), which was similar to that of calmodulin. The selectivity of CBP for metal ion binding was compared among Ca(2+), Cu(2+), and Zn(2+). The binding constant was obtained through a similar competitive binding study with murexide. The binding constants for Ca(2+), Cu(2+), and Zn(2+) were 7.0 × 10(5), 4.2 × 10(5), and 1.7 × 10(5) M(-1), respectively, indicating 2-4-fold higher selectivity of CBP for Ca(2+) compared to Cu(2+) and Zn(2+). The CBP has selectivity for Ca(2+), and binding affinity for Ca(2+) was similar to the biological Ca(2+) binding motif of calmodulin.
authors.affiliation: Department of Chemistry and Nanoscience, Ewha Womans University , 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea.
authors.forename: Hee Jung
authors.initials: HJ
authors.lastname: Chung
authors.affiliation: Department of Chemistry and Nanoscience, Ewha Womans University , 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea.
authors.forename: Du Young
authors.initials: du Y
authors.lastname: Ko
authors.affiliation: Department of Chemistry and Nanoscience, Ewha Womans University , 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea.
authors.forename: Hyo Jung
authors.initials: HJ
authors.lastname: Moon
authors.affiliation: Department of Chemistry and Nanoscience, Ewha Womans University , 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea.
authors.forename: Byeongmoon
authors.initials: B
authors.lastname: Jeong
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D020832:EF Hand Motifs; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism
chemical_list: D002147:Calmodulin; D010455:Peptides; D003300:Copper; D015032:Zinc; D002118:Calcium