铽离子-镧系金属结合标签的荧光探针的改进
收稿日期: 2015-04-13
修回日期: 2016-01-26
网络出版日期: 2016-03-05
基金资助
国家自然科学基金青年科学基金资助项目(31400735).
Improvement of Fluorescence Quantum Yield for Terbium (III)-Bound Lanthanide-Binding Tag
Received date: 2015-04-13
Revised date: 2016-01-26
Online published: 2016-03-05
镧系金属因其具有较窄的发射光谱带,较大的斯托克斯位移以及毫秒级的荧光寿命,而被广泛应用于荧光检测中,其中Tb3+、Eu3+最为常用.镧系金属结合标签(Lanthanide Binding Tag,LBT)可以与蛋白质融合表达,并且一般不会影响蛋白的结构和功能,这些特点使LBT被广泛应用于蛋白质结构与功能研究中.LBT能够特异性地结合镧系金属离子,并利用LBT上色氨酸的吲哚环作为“天线”吸收外部能量,再将能量传递给镧系金属离子,进而激发镧系金属离子产生荧光.该文以经典模式蛋白泛素(Ubiquitin,Ub)作为媒介,将LBT引入到Ub的碳端,采用定点突变的方法增加LBT上吲哚环的数量,观察Ub-LBT [结合铽离子(Tb3+)]荧光量子产率的变化.结果表明在LBT结构中增加吲哚环的数量能够提高LBT(结合Tb3+)的荧光量子产率.
关键词： 荧光光谱学; 镧系金属结合标签(LBT); 荧光量子产率; 解离常数; 泛素(Ub)
陈少敏 , 唐淳 , 龚洲 . 铽离子-镧系金属结合标签的荧光探针的改进[J]. 波谱学杂志, 2016 , 33(1) : 106 -116 . DOI: 10.11938/cjmr20160110
Long-lived luminescence lifetime and large Stokes shift of the lanthanide ion made it widely used in fluorescence detection. The lanthanide-binding tag (LBT) is a small, genetically encoded, versatile protein fusion partner that specifically binds a lanthanide ion with high affinity (KD ≈10-9 mol/L). These properties make LBT wildly used for studies on protein structures and functions. Lanthanide in LBT cannot be excited directly, and tryptophan is needed to serves as a sensitizer to absorb energy and transfer energy to the lanthanide. We introduced LBT onto the C-terminal of ubiquitin (Ub), and increased the number of indole rings in LBT using site-directed mutagenesis to increase the quantum yield of Tb (III)-bound Ub-LBT.
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