Martin Langdon J, Imperiali Barbara
Department of Chemistry and Physics, Warren Wilson College, Asheville, NC, USA.
Methods Mol Biol. 2015;1248:201-20. doi: 10.1007/978-1-4939-2020-4_14.
Consider the lanthanide metals, comprising lanthanum through lutetium. Lanthanides form stable cations with a +3 charge, and these ions exhibit a variety of useful physical properties (long-lifetime luminescence, paramagnetism, anomalous X-ray scattering) that are amenable to studies of biomolecules. The absence of lanthanide ions in living systems means that background signals are generally a nonissue; however, to exploit the advantageous properties it is necessary to engineer a robust lanthanide-binding sequence that can be appended to any macromolecules of interest. To this end, the luminescence produced by tryptophan-sensitized Tb(3+) has been used as a selection marker for peptide sequences that avidly chelate these ions. A combinatorial split-and-pool library that uses two orthogonal linkers-one that is cleaved for selection and one that is cleaved for sequencing and characterization-has been used to develop lanthanide-binding tags (LBTs): peptides of 15-20 amino acids with low-nM affinity for Tb(3+). Further validating the success of this screen, knowledge about LBTs has enabled the introduction of a lanthanide-binding loop in place of one of the four native calcium-binding loops within the protein calcineurin B.
考虑镧系金属，包括从镧到镥的元素。镧系元素形成带 +3 电荷的稳定阳离子，这些离子展现出多种有用的物理性质（长寿命发光、顺磁性、反常 X 射线散射），适用于生物分子研究。生物系统中不存在镧系离子意味着背景信号通常不成问题；然而，为了利用这些有利性质，有必要设计一种能与任何感兴趣的大分子相连的强大镧系元素结合序列。为此，色氨酸敏化的 Tb(3+) 产生的发光已被用作对这些离子有强烈螯合作用的肽序列的选择标记。一种使用两个正交连接子的组合拆分 - 混合文库——一个用于选择时切割，另一个用于测序和表征时切割——已被用于开发镧系元素结合标签（LBTs）：对 Tb(3+) 具有低纳摩尔亲和力的 15 - 20 个氨基酸的肽。进一步验证该筛选的成功，关于 LBTs 的知识使得能够在蛋白质钙调神经磷酸酶 B 的四个天然钙结合环之一的位置引入一个镧系元素结合环。
