Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids.
Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids.
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DOI：
10.1002/anie.201308794
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发表时间：
2014-02-17
期刊：
影响因子：
--
通讯作者：
Wang L
中科院分区：
文献类型：
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作者：
Xiang Z;Lacey VK;Ren H;Xu J;Burban DJ;Jennings PA;Wang L
蛋白质之间和内部共价键的选择性生成将为研究蛋白质功能和工程化具有新特性的蛋白质提供新途径。通过使非天然氨基酸 (Uaa) 选择性地与邻近的天然残基反应，将新的共价键通过基因引入蛋白质中。这种接近启用的生物反应性已扩展到一系列卤代烷 Uaas。正交的 tRNA/合成酶对被进化来整合这些 Uaas，它们仅在靠近时才与半胱氨酸形成共价硫醚键。通过使用 Uaa 和半胱氨酸，在亲和体与其底物 Z 蛋白之间证明了自发的共价键形成，从而导致不可逆的结合，并且在亲和体内增加了其热稳定性。这种接近启用的蛋白质交联（PEPC）策略通常可以扩展到针对不同的天然氨基酸，从而为蛋白质研究和蛋白质工程提供共价键形成的多样性和灵活性。
The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity-enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity-enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering.