Noncanonical amino acids in the interrogation of cellular protein synthesis.
Noncanonical amino acids in the interrogation of cellular protein synthesis.
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DOI：
10.1021/ar200144y
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发表时间：
2011-09-20
影响因子：
18.3
通讯作者：
Tirrell, David A.
中科院分区：
文献类型：
--
作者：
Ngo, John T.;Tirrell, David A.
活细胞中的蛋白质可以通过用适当设计的非规范氨基酸（ncAA）进行代谢标记来接受生物正交化学。在最简单的代谢标记方法中，氨基酸类似物取代了感兴趣的蛋白质基因（或多个基因）指定的天然氨基酸之一。通过控制实验条件，可以调节置换的程度。这种方法，通常称为残基特异性掺入，允许ncAA以受控比例掺入天然氨基酸残基通常占据的位置。对于以这种方式用ncAA标记的蛋白质，它必须满足两个要求：（i）相应的天然氨基酸必须在基因水平上编码在蛋白质序列内，以及（ii）蛋白质必须在ncAA在细胞中时表达。由于这种方法允许在整个细胞中标记蛋白质，因此它使我们能够开发通过用反应性ncAA标记蛋白质来跟踪细胞蛋白质合成的策略。在类似于同位素标记的程序中，在新合成的蛋白质被标记的“脉冲”期间，将具有抑制活性的ncAA掺入蛋白质中。通过将ncAA侧链生物正交地连接到允许检测、分离和可视化标记的蛋白质的探针，可以将标记的蛋白质组与脉冲之前制备的那些区分开。侧链含有叠氮基、炔基或烯基的非规范氨基酸在这类实验中特别有用。它们以蛋氨酸类似物的形式被掺入蛋白质中，蛋氨酸类似物是天然翻译机制的底物。该方法的选择性可以通过使用突变氨酰转移RNA合成酶（aaRS）来增强，所述突变氨酰转移RNA合成酶（aaRS）允许掺入内源性生物机械不使用的ncAA。通过突变aaRS的表达，蛋白质可以用其他有用的ncAA标记，包括含有酮或芳基卤化物的类似物。高通量筛选策略可以鉴定激活广泛ncAA的阿尔斯变体。突变体合成酶的受控表达已与ncAA标记相结合，以允许蛋白质的细胞选择性代谢标记。在复杂细胞混合物内的一部分细胞中突变合成酶的表达限制了对该细胞亚群的标记。在不表达合成酶的细胞中合成的蛋白质既不被标记也不被检测。在多细胞环境中，这种方法允许识别标记蛋白质的细胞来源。在这个帐户中，我们总结了已经开发的工具和策略，通过残基特异性标记与ncAA的询问细胞蛋白质合成。我们描述了ncAA标记策略的化学和遗传组成部分，并讨论了这些方法是如何被用于化学生物学。
Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed, non-canonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein’s gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a “pulse” in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Non-canonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl transfer RNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.
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影响因子：
46.2
作者：
Jewett JC;Bertozzi CR
通讯作者：
Bertozzi CR
影响因子：
15
作者：
Brustad EM;Lemke EA;Schultz PG;Deniz AA
通讯作者：
Deniz AA
影响因子：
3.2
作者：
Beatty, Kimberly E.;Fisk, John D.;Smart, Brian P.;Lu, Ying Ying;Szychowski, Janek;Hangauer, Matthew J.;Baskin, Jeremy M.;Bertozzi, Carolyn R.;Tirrell, David A.
通讯作者：
Tirrell, David A.
DOI：
10.1073/pnas.0808480105
发表时间：
2008-10-14
影响因子：
11.1
作者：
Jao, Cindy Y.;Salic, Adrian
通讯作者：
Salic, Adrian
影响因子：
3.7
作者：
Cappella, Paolo;Gasparri, Fabio;Moll, Juergen
通讯作者：
Moll, Juergen