Lanthanoid tagging via an unnatural amino acid for protein structure characterization
Lanthanoid tagging via an unnatural amino acid for protein structure characterization
复制标题
通过非天然氨基酸进行镧系元素标记，用于蛋白质结构表征
DOI：
10.1007/s10858-017-0106-9
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发表时间：
2017-04-01
影响因子：
2.7
通讯作者：
Tang, Chun
中科院分区：
文献类型：
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作者：
Jiang, Wen-Xue;Gu, Xin-Hua;Tang, Chun
镧系拟接触位移(PCS)提供了顺磁标记和蛋白质核之间的长程结构信息。然而，对于带有天然半胱氨酸的蛋白质，特定位点的结合可能只利用与巯基化学垂直的官能团。在这里，我们报道了两个稀土探针，DTTA-C3-yne和DTTA-C4-yne，它们可以通过叠氮-炔环加成反应与靶蛋白中的非天然氨基酸pAzF偶联。通过泛素和含半胱氨酸酶EIIB的验证，我们发现对于每个标记/稀土元素组合，都可以产生不同特征的大的PCS。由于标签的刚性，基于DTTA的稀土标签与大的磁化率张量有关。特别是，DTTA-C3标签的引入提供了分子间PCSS，并使泛素和UBA结构域之间的瞬时蛋白质复合体的结构表征成为可能。我们一起扩展了顺磁标签的曲目和顺磁核磁共振的适用性。
Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.