Double-lanthanide-binding tags:: Design, photophysical properties, and NMR applications
Double-lanthanide-binding tags:: Design, photophysical properties, and NMR applications
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DOI：
10.1021/ja070480v
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发表时间：
2007-06-06
影响因子：
15
通讯作者：
Imperiali, Barbara
中科院分区：
文献类型：
--
作者：
Martin, Langdon J.;Haehnke, Martin J.;Imperiali, Barbara
镧系元素结合标记(LBT)是由20个编码氨基酸组成的多肽序列，可以紧密和选择性地络合稀土离子，并能敏化Tb(Tb3+)发光。在这些性质的基础上，预测增加结合的稀土元素的数量将提高这些标签的性能。因此，以结构特征良好的单LBT序列为起点，设计了连接两个稀土元素结合基序的双LBT(DLBT)。在这里，我们报道了dLBT多肽的产生以及dLBT标记的泛素融合蛋白的发光和核磁共振研究。这些与稀土元素结合的结构被证明是改进的发光标签，具有活跃的稀土结合和高达3倍的发光强度。在泛素结构上进行了核磁共振实验，其中结合的顺磁性稀土元素作为取向诱导剂获得了剩余的偶极偶联，这是确定大分子结构的有价值的限制条件。总之，这些结果表明，dLBTS将成为生物物理应用的有价值的化学工具，从而为研究蛋白质的结构、功能和动力学提供新的方法。
Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.