Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral
Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral
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DOI：
10.1073/pnas.97.22.11984
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发表时间：
2000-10-24
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Baird, GS;Zacharias, DA;Tsien, RY
DsRed是一种近期克隆出的28 - kDa荧光蛋白，它是导致盘形海葵属（Discosoma - genus）珊瑚口盘周围呈红色的原因。作为一种潜在的表达示踪剂和融合伴侣，DsRed引起了极大的关注，它可与来自水母（Aequorea）的同源绿色荧光蛋白互补，但人们对DsRed的生物化学性质知之甚少。我们现在发现，DsRed的消光系数和量子产率比之前报道的要高得多，并且对极端pH值和光漂白具有极好的耐受性。此外，通过将第83位的赖氨酸突变为甲硫氨酸，其最大发射波长可从583 nm进一步移至602 nm。然而，DsRed存在重大缺陷，例如强烈的寡聚化和缓慢的成熟过程。分析超速离心证明DsRed在体外是一种强制性的四聚体，荧光共振能量转移测量和酵母双杂交试验证实了其在活细胞中的寡聚化。而且，DsRed在体外或体内从绿色完全成熟为红色需要数天时间，像第83位赖氨酸突变为精氨酸这样的突变会阻止颜色变化。DsRed在细胞生物学上的许多潜在应用都需要抑制其四聚化并加速其成熟过程。
DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma- genus, DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.