Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it.
Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it.
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DOI：
10.1038/srep22342
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发表时间：
2016-03-09
影响因子：
4.6
通讯作者：
Watanabe TM
中科院分区：
文献类型：
--
作者：
Morikawa TJ;Fujita H;Kitamura A;Horio T;Yamamoto J;Kinjo M;Sasaki A;Machiyama H;Yoshizawa K;Ichimura T;Imada K;Nagai T;Watanabe TM
荧光蛋白由于其在活体标本中的相容性和多种应用而在生物学中得到广泛应用。荧光蛋白通常对细胞内条件如pH和离子浓度不期望地敏感，有时产生相当大的问题。然而，利用这些固有的灵敏度可以帮助开发功能探针。在这项研究中，我们发现黄色荧光蛋白（YFP）的荧光依赖于溶液中的蛋白质浓度，这种依赖性可以通过在YFP中添加甘氨酸残基来增强;我们将这一发现应用于构建细胞内蛋白质拥挤传感器。Förster共振能量转移（FRET）对，涉及对蛋白质浓度不敏感的青色荧光蛋白（CFP）和甘氨酸插入的YFP，作为遗传编码的探针来评估细胞内拥挤。通过测量本FRET探针的荧光，我们能够检测活细胞中蛋白质聚集的动态变化。
Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Förster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.