Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.
Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.
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DOI：
10.1016/j.bmc.2016.07.008
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发表时间：
2016-09-15
影响因子：
3.5
通讯作者：
Hecht SM
中科院分区：
文献类型：
--
作者：
Talukder P;Dedkova LM;Ellington AD;Yakovchuk P;Lim J;Anslyn EV;Hecht SM
与核酸结合并调节其结构和功能的蛋白质数量众多，并且非常重要。这些蛋白质采用多种策略来识别其核酸底物中的相关结构元件，其中一些已被证明涉及相当微妙的相互作用，这可能很难从第一原理设计。在本研究中，我们探索了含有非天然氨基酸的蛋白质的制备，这些非天然氨基酸具有核碱基侧链。原则上，将多个核碱基氨基酸引入蛋白质的核酸结合结构域应使这些修饰的蛋白质能够使用沃森-克里克碱基配对相互作用与其核酸底物相互作用。我们描述了五个丙氨酰核碱基氨基酸的合成保护的方式，使他们的附件的抑制tRNA，并将其纳入两种蛋白质的每一个可接受的效率。所研究的核碱基包括胞嘧啶、尿嘧啶、胸腺嘧啶、腺嘌呤和鸟嘌呤，即DNA和RNA的主要核碱基成分。选择二氢叶酸还原酶作为一种模型蛋白质，以能够直接比较核碱基氨基酸与先前研究的许多其他非天然氨基酸的掺入能力。选择DNA聚合酶I的Klenow片段作为代表性的DNA结合蛋白，其作用模式已被详细研究。
Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.