Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University , Tempe, Arizona 85287, United States.
J Am Chem Soc. 2017 Oct 11;139(40):14098-14108. doi: 10.1021/jacs.7b05168. Epub 2017 Sep 27.
Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site-directed mutagenesis and expressed in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-o-nitrobenzyl protection of tyrosine phosphate enabled its incorporation into DHFR and IκB-α using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in parallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNA by UAG codon suppression during in vitro translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600-2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600-2605 and incorporated unprotected phosphotyrosine into IκB-α using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-κB binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated IκB-α also mediated the exchange of exogenous DNA into an NF-κB-cellular DNA complex isolated from the nucleus of activated Jurkat cells.
磷酸化蛋白质在许多不同的细胞网络调节中发挥着重要作用。然而，与制备含有未修饰的蛋白质氨基酸的蛋白质不同，这些蛋白质可以通过定点突变轻易改变，并在体外和体内表达，制备在预定位置磷酸化的蛋白质不能轻易且以可接受的产率完成。为了能够合成用于体外研究的磷酸化蛋白质，我们探索了使用带有化学保护基团的磷酸化氨基酸，从而消除了已被证明对蛋白质翻译有负面影响的负电荷。酪氨酸磷酸盐的双邻硝基苄基保护使其能够使用野生型核糖体掺入 DHFR 和 IκB-α中，并且可以通过光解来脱保护所合成的蛋白质。同时还平行研究了大肠杆菌 23S rRNA 的重新设计，通过使用磷酸化的嘌呤霉素来指导，以鉴定能够通过 UAG 密码子抑制在体外翻译期间将未保护的磷酸酪氨酸掺入蛋白质中的修饰核糖体。用磷酸化嘌呤霉素选择修饰核糖体的文库，鉴定了在 23S rRNA 的核苷酸 2600-2605 处具有突变的六个修饰核糖体变体；这些变体对磷酸化嘌呤霉素的敏感性增强。这六个克隆在 2600-2605 区域具有一些序列同源性，并使用在对应于蛋白质第 42 位氨基酸的位置处具有 TAG 密码子的修饰基因将未保护的磷酸酪氨酸掺入 IκB-α中。纯化的磷酸化蛋白质与磷酸酪氨酸特异性抗体结合，并允许 NF-κB 与对应于其在 IL-2 基因启动子中结合位点的 DNA 双链序列结合。出乎意料的是，磷酸化的 IκB-α还介导将外源性 DNA 交换到从激活的 Jurkat 细胞的核中分离的 NF-κB-细胞 DNA 复合物中。
