GFP Loss-of-Function Mutations in Arabidopsis thaliana
GFP Loss-of-Function Mutations in Arabidopsis thaliana
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DOI：
10.1534/g3.115.019604
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发表时间：
2015-09-01
影响因子：
2.6
通讯作者：
Matzke, Marjori
中科院分区：
文献类型：
--
作者：
Fu, Jason L.;Kanno, Tatsuo;Matzke, Marjori
绿色荧光蛋白（GFP）及其相关荧光蛋白在生物学研究中被广泛用于监测活细胞中的基因表达和蛋白质定位。GFP发色团在氧气存在下通过涉及内部三肽Ser 65（或Thr 65）-Tyr 66-Gly 67的环化的多步反应自发产生，所述内部三肽嵌入在11链β-桶结构的中心。随机和位点特异性诱变已被用于优化GFP荧光并产生具有新特性的衍生物。然而，有助于理解GFP蛋白折叠和发色团形成的功能丧失突变尚未完全编目。在这里，我们报告了一个收集的甲基磺酸乙酯诱导的GFP功能丧失突变的模式植物拟南芥。改变对发色团成熟重要的残基的突变，如Arg 96和Ser 205，大大减少或消除荧光，而不显著改变GFP蛋白的积累。相比之下，其他荧光缺失突变大大减少了GFP蛋白的量，表明它们损害了蛋白质的稳定性。这一类中的许多突变产生高度保守的甘氨酸残基的取代，包括以下：显色三肽中的Gly 67;第二β链中的Gly 31、Gly 33和Gly 35;以及β桶支架盖中的Gly 20、Gly 91和Gly 127。我们的遗传分析支持结构和生化研究的结论，并证明了多个高度保守的甘氨酸残基在GFP蛋白稳定性中的关键作用。
Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded beta-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate-induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second beta-strand; and Gly20, Gly91, and Gly127 in the lids of the beta-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability.