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.

Multiple Evolutionary Origins of Ubiquitous Cu2+ and Zn2+ Binding in the S100 Protein Family

“…For example, Gribenko et al proposed that human S100P may bind Zn 2+ in one of the Ca 2+ binding motifs [ 75 ]. EF-hands often discriminate Ca 2+ from Zn 2+ and Cu 2+ , however, so this likely does not explain all of the observed transition metal binding [ 33 , 76 – 78 ].…”

Self-Correcting Method for the Measurement of Free Calcium and Magnesium Concentrations by 1H NMR

“…4,5 The free ion concentrations can be measured using electrochemical probes [5][6][7] or fluorescent dyes. [8][9][10] However, such measurements can be expensive and inconvenient to perform in addition to NMR, particularly when only limited quantities of sample are available. Furthermore, the insertion of electrodes can cause serious damage to soft-solid samples such as gels while electrochemical methods are also wholly unsuited to study the internal ion gradients that can occur in these systems.…”

“…When complexing species are present, the concentration of free ions can be very different to the total concentrations of Ca or Mg. The free ion concentrations thus grant valuable insight into the ion-responsive behavior of the systems. , The free ion concentrations can be measured using electrochemical probes − or fluorescent dyes. − However, such measurements can be expensive and inconvenient to perform in addition to NMR, particularly when only limited quantities of sample are available. Furthermore, the insertion of electrodes can cause serious damage to soft-solid samples such as gels while electrochemical methods are also wholly unsuited to study the internal ion gradients that can occur in these systems. , Methods for the direct measurement of free Ca 2+ and Mg 2+ concentrations by NMR are thus required.…”

Recent progress in luminescent self-healing gels

“…这些功能更全面、安全性能 更高的多功能、 智能柔性凝胶材料能够通过调节自身 的性质适应外界刺激变化, 并且可使凝胶材料的多 种功能发挥协同作用, 显著推动了自愈合凝胶的基 础研究, 拓展了其应用潜能. 其中, 既具有自愈性能 又具有发光性能的发光自愈合凝胶, 作为一种新型 的 功 能 性 自 愈 合 凝 胶 , 因 集 成 发 光 凝 胶 在 生 物 成 像 [37,38] 、光学开关 [39] 、生物检测 [40,41] 、温度检测 [42,43] 、 pH [44,45] 响应等方面的优势具有广泛潜在应用而受到 关注. 发光凝胶利用其发光性, 能监测甚至响应体内 的生理物质变化, 也可进行原位跟踪药物释放, 便于 其在实际使用过程中的无损实时监控和检测 [41,46] .…”