A Bifunctional Noncanonical Amino Acid: Synthesis, Expression, and Residue-Specific Proteome-wide Incorporation,Biochemistry

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A Bifunctional Noncanonical Amino Acid: Synthesis, Expression, and Residue-Specific Proteome-wide Incorporation
Biochemistry ( IF 2.9 ) Pub Date : 2018-06-22 00:00:00 , DOI: 10.1021/acs.biochem.8b00397
Jan-Erik Hoffmann ₁ , Dmytro Dziuba ₂ , Frank Stein ₂ , Carsten Schultz _{1,

2}

Affiliation

Mapping of weak and hence transient interactions between low-abundance interacting molecules is still a major challenge in systems biology and protein biochemistry. Therefore, additional system-wide acting tools are needed to determine protein interactomics. Most important are reagents that can be applied at any kind of protein interface and the possibility to enrich cross-linked fragments with high efficiency. In this study, we report the synthesis of a novel noncanonical amino acid that features a diazirine group for ultraviolet cross-linking as well as an alkyne group for labeling by click chemistry. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal structural perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell cultures, and we show that PrDiAzK allows protein labeling as well as cross-linking. In addition, we tested PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation, implying potential applications in system-wide mapping of protein–protein interactions in the future.

中文翻译：

双功能非规范氨基酸：合成，表达和特定于蛋白质组的残留。

低丰度相互作用分子之间的弱相互作用的映射和因此的瞬时相互作用仍然是系统生物学和蛋白质生物化学中的主要挑战。因此，需要其他系统范围内的作用工具来确定蛋白质相互作用组学。最重要的是可以应用于任何类型蛋白质界面的试剂，以及高效富集交联片段的可能性。在这项研究中，我们报告了一种新颖的非规范性氨基酸的合成，该特征具有用于紫外线交联的重氮基以及通过点击化学进行标记的炔基。这种双功能氨基酸，称为PrDiAzK，可以使用遗传密码扩展法以最小的结构扰动插入几乎任何蛋白质界面。我们证明可以在细菌和哺乳动物细胞培养物中将PrDiAzK选择性结合到蛋白质中，并且我们证明PrDiAzK可以进行蛋白质标记以及交联。此外，我们通过翻译的随机正交编码测试了PrDiAzK在蛋白质组范围内的整合，这暗示了将来在蛋白质-蛋白质相互作用的系统范围内的潜在应用。

更新日期：2018-06-22

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