FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule
FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule
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DOI：
10.1038/nsb728
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发表时间：
2001-12-01
期刊：
影响因子：
--
通讯作者：
Ikura, M
中科院分区：
文献类型：
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作者：
Truong, K;Sawano, A;Ikura, M
细胞内Ca ~（2+）作为第二信使，调节细胞的发育、分化和凋亡等多种生理现象。Cameleon是一类基于绿色荧光蛋白（GFP）和钙调素（CaM）的钙离子荧光指示剂，已被证明是测量活细胞中游离钙离子浓度的有用工具。然而，传统的骆驼有一个小的动态范围的荧光共振能量转移（FRET），使Ca 2+浓度的微妙变化难以检测和研究在一些细胞和细胞器。使用NMR结构的钙调素绑定到钙调素结合肽来自钙调素依赖性激酶激酶（CKKP），我们已经合理地设计了一个新的cameleon，显示出两倍的增加，在FRET动态范围内的生理意义范围内的细胞质Ca 2+浓度为0.05-1 μ M。
Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a twofold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 muM.