铽离子-镧系金属结合标签的荧光探针的改进

Improvement of Fluorescence Quantum Yield for Terbium(Ⅲ)-Bound Lanthanide-Binding Tag

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Tb^(3+)、Eu^(3+)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(Tb^(3+))]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Tb^(3+))的荧光量子产率.

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.