Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins
Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins
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DOI：
10.1073/pnas.0400417101
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发表时间：
2004-07-20
影响因子：
11.1
通讯作者：
Miyawaki, A
中科院分区：
文献类型：
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作者：
Nagai, T;Yamada, S;Miyawaki, A
荧光共振能量转移（FRET）技术已被用于开发各种细胞功能的遗传编码荧光指示器。虽然大多数指示器都有发出青色和黄色的荧光蛋白（CFP和YFP）作为FRET供体和受体，但它们较差的动态范围常常妨碍检测细微但重要的信号。在这里，我们通过不同角度的YFP融合，优化了Ca2+指示剂黄色cameleon （YC）中两个发色团的相对取向。我们生成了循环排列的yfp (cpyfp)，显示出有效的成熟和酸稳定性。其中一个加入到YC中的cpYFPs从Ca2+饱和形式的CFP中吸收了大量的激发能量，从而使YFP/CFP的Ca2+依赖性变化增加了近600%。在培养细胞和转基因小鼠的神经系统中，新的YC能够以比以前更好的空间和时间分辨率可视化亚细胞Ca2+动态。我们的研究为基于gfp的FRET指标的开发和改进提供了重要的指导。
Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca2+ indicator, yellow cameleon (YC) by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca2+-saturated form, thereby increasing the Ca2+-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca2+ dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.