Single-site glycine-specific labeling of proteins
Single-site glycine-specific labeling of proteins
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DOI：
10.1038/s41467-019-10503-7
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发表时间：
2019-06-10
影响因子：
16.6
通讯作者：
Rai, Vishal
中科院分区：
文献类型：
--
作者：
Purushottam, Landa;Adusumalli, Srinivasa Rao;Rai, Vishal
天然蛋白质的标记引起了科学各个领域的兴趣。然而，在蛋白质的单个位点进行精确标记仍然存在重大的未解决的挑战。在这里，我们报告了蛋白质中天然或易于设计的 N 末端甘氨酸的位点特异性标记，具有显着的效率和选择性。该方法从 N 末端亚胺产生潜在亲核试剂，在生理条件下与醛反应生成氨基醇。它将 N-Gly 区分为其他蛋白氨基酸中的独特靶点。该方法允许对分离形式的蛋白质进行单位点标记，并延伸至裂解的细胞。它管理一个正交醛基，为后期标记亲和标签、F-19 NMR 探针和荧光团做好准备。用户友好的方案可提供分析纯的标记蛋白质。温和的反应条件不会改变蛋白质的结构和功能。荧光团标记的胰岛素的细胞摄取及其激活胰岛素受体介导的信号传导的能力保持不变。
Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, F-19 NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.