Genetically Encoding Photocaged Quinone Methide to Multitarget Protein Residues Covalently in Vivo
Genetically Encoding Photocaged Quinone Methide to Multitarget Protein Residues Covalently in Vivo
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DOI：
10.1021/jacs.9b01738
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发表时间：
2019-06-19
影响因子：
15
通讯作者：
Wang, Lei
中科院分区：
文献类型：
--
作者：
Liu, Jun;Li, Shanshan;Wang, Lei
通过基因将共价键引入具有残基特异性的蛋白质中，为蛋白质研究和工程提供了创新的途径，但迄今为止，基因编码的潜在生物反应性非天然氨基酸（Uaas）仅与一到几种天然残基发生反应，限制了蛋白质的种类和适用于该技术的应用范围。本文报道了大肠杆菌和哺乳动物细胞中（2R)-2-氨基-3-氟-3-（4-（2-硝基苯基）氧）苯基）丙酸（FnbY）的遗传编码。在光激活后，FnbY产生了一种活性的甲基醌（QM），它直接与活细胞中蛋白质附近的9个天然氨基酸残基选择性反应。除了Cys、Lys、His和Tyr外，光活化的FnbY还能与Trp、Met、Arg、Asn和Gln发生反应，这些都是现有潜伏性生物反应性waas无法达到的。因此，FnbY极大地增加了体内共价靶向的残基数量。QM的半衰期较传统光交联uaa中间体长，FnbY的交联效率高于对叠氮基苯丙氨酸。FnbY对亲核残基具有选择性的光激活性和多靶点反应性，将对处理多种蛋白质和通过利用体内共价键在化学生物学、生物治疗学和蛋白质工程中扩大应用范围具有价值。
Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in Escherichia coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His, and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn, and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-cross-linking Uaas, and FnbY exhibited crosslinking efficiency higher than p-azido-phenylalanine. The photoactivatable and multitargeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.