Ozawa T, Kakuta M, Sugawara M, Umezawa Y, Ikura M
Department of Chemistry, School of Science, University of Tokyo, Japan.
Anal Chem. 1997 Aug 1;69(15):3081-5. doi: 10.1021/ac9613141.
A method for evaluating a physiologically relevant ion selectivity of Ca2+ signaling pathways in biological cells based on a Ca(2+)-dependent on/off switch for cellular processes via calmodulin (CaM) chemistry is described. CaM serves as a primary ion receptor for Ca2+ and a given CaM-binding peptide as a target for a CaM-Ca2+ complex. Upon accommodating four Ca2+ ions in its binding sites, CaM undergoes a conformational change to form a CaM-Ca(2+)-target peptide ternary complex. This Ca(2+)-induced selective binding of the Ca(2+)-CaM complex to the target peptide was monitored by a surface plasmon resonance (SPR) technique. As a target peptide, a 26-amino acid residue of M13 derived from skeletal muscle myosin light-chain kinase was used. The target peptide was covalently immobilized in the dextran matrix on top of gold, over which sample solutions containing Ca2+ and CaM were injected in a flow system. Ca(2+)-dependent SPR signals were observed for Ca2+ concentrations from 3.2 x 10(-8) to 1.1 x 10(-5) M and it leveled off. The observed SPR signals were explained as due to an increase in the refractive indexes caused by a Ca2+ ion-switched protein/ peptide interaction, i.e., Ca2+ ion to CaM and subsequent additional binding of the thus formed complex with immobilized M13. No SPR signals were however, induced by Mg2+, K+, and Li+ at concentrations as high as 1.0 x 10(-1) M; these results and previous spectroscopic data taken together conclude that these ions do not induce CaM/peptide interaction. Large changes in SPR signals were observed with a Sr2+ ion concentration over 5.1 x 10(-4) M; Sr2+ ion behaved in this case as a strong agonist toward the Ca(2+)-dependent on/off switch of CaM. The present system thus exhibited "physiologically more relevant" ion selectivity in that relevant metal ions could switch on the CaM/peptide or -protein interaction rather than merely be bound to CaM causing no further signal transduction. The potential use of this finding for more widely evaluating cation selectivity toward the Ca2+ signaling process was discussed.
描述了一种基于通过钙调蛋白（CaM）化学实现细胞过程的Ca（2 +）依赖性开/关开关来评估生物细胞中Ca2 +信号通路的生理相关离子选择性的方法。CaM作为Ca2 +的主要离子受体，给定的CaM结合肽作为CaM-Ca2 +复合物的靶标。在其结合位点容纳四个Ca2 +离子后，CaM发生构象变化以形成CaM-Ca（2 +）-靶肽三元复合物。通过表面等离子体共振（SPR）技术监测Ca（2 +）诱导的Ca（2 +）-CaM复合物与靶肽的选择性结合。作为靶肽，使用了源自骨骼肌肌球蛋白轻链激酶的26个氨基酸残基的M13。靶肽共价固定在金表面的葡聚糖基质中，在流动系统中注入含有Ca2 +和CaM的样品溶液。对于3.2×10（-8）至1.1×10（-5）M的Ca2 +浓度观察到Ca（2 +）依赖性SPR信号，并且信号趋于平稳。观察到的SPR信号被解释为是由于Ca2 +离子切换的蛋白质/肽相互作用引起的折射率增加，即Ca2 +离子与CaM结合，随后形成的复合物与固定化的M13进一步结合。然而，在高达1.0×10（-1）M的浓度下，Mg2 +、K +和Li +不会诱导SPR信号；这些结果与先前的光谱数据一起得出结论，这些离子不会诱导CaM /肽相互作用。当Sr2 +离子浓度超过5.1×10（-4）M时，观察到SPR信号有很大变化；在这种情况下，Sr2 +离子作为CaM的Ca（2 +）依赖性开/关开关的强激动剂。因此，本系统表现出“生理上更相关”的离子选择性，因为相关金属离子可以开启CaM /肽或 - 蛋白质相互作用，而不仅仅是与CaM结合而不引起进一步的信号转导。讨论了这一发现对于更广泛评估阳离子对Ca2 +信号传导过程的选择性的潜在用途。
