Novel fluorescent indicator proteins for monitoring free intracellular Ca2+
Novel fluorescent indicator proteins for monitoring free intracellular Ca2+
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DOI：
10.1016/s0143-4160(97)90014-2
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发表时间：
1997-09-01
期刊：
影响因子：
4
通讯作者：
Romoser, VA
中科院分区：
文献类型：
--
作者：
Persechini, A;Lynch, JA;Romoser, VA
我们最近描述了一种荧光指示剂蛋白，其中绿色荧光蛋白的红移和蓝移变体与来自平滑肌肌球蛋白轻链激酶的钙调蛋白结合序列连接[Romoser V.A.、Hinkle P.M.、Persechini A.在活细胞中检测由钙调蛋白结合序列连接的两个绿色荧光蛋白变体组成的指示剂荧光的 Ca2+依赖性变化。一类新的荧光指示剂。生物化学杂志1997； 272：13270-13274]。当 (Ca2+)(4)-钙调蛋白结合时，该蛋白质在 505 nm（380 nm 激发）处的荧光发射减少了约 65%，而在 440 nm 处的荧光发射则成比例增加。我们发现，工程化钙调蛋白（其中 C 端和 N 端 EF 手对已交换）与该蛋白质的 C 端融合，产生了一种新的指示剂，可直接响应 Ca2+ 离子浓度的变化，在 0.5 mM Mg2+ 存在的情况下，Ca2+ 的表观 K-d 值为 100 nM。通过改变钙调蛋白结合序列的氨基酸序列，减弱其与内在钙调蛋白结构域的相互作用，可以降低指示剂对 Ca2+ 的亲和力。该指示剂的荧光发射可用于监测活细胞内游离Ca2+离子浓度的生理变化。
We have recently described a fluorescent indicator protein in which red-and blue-shifted variants of green fluorescent protein are joined by the calmodulin-binding sequence from smooth muscle myosin light chain kinase [Romoser V.A., Hinkle P.M., Persechini A. Detection in living cells of Ca2+-dependent changes in the fluorescence of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 1997; 272: 13270-13274]. The fluorescence emission of this protein at 505 nm (380 nm excitation) is reduced by similar to 65% when (Ca2+)(4)-calmodulin is bound, with a proportional increase in fluorescence emission at 440 nm. We have found that fusion of an engineered calmodulin, in which the C- and N-terminal EF hand pairs have been exchanged, to the C-terminus of this protein results in a novel indicator that responds directly to changes in the Ca2+ ion concentration, with an apparent K-d value of 100 nM for Ca2+ in the presence of 0.5 mM Mg2+. The affinity of the indicator for Ca2+ can be decreased by altering the amino acid sequence of the calmodulin-binding sequence to weaken its interaction with the intrinsic calmodulin domain. The fluorescence emission of this indicator can be used to monitor physiological changes in the free Ca2+ ion concentration in living cells.