Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing.
Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing.
复制标题
DOI：
10.1038/nmeth.4302
复制
发表时间：
2017-07
期刊：
影响因子：
48
通讯作者：
Chin JW
中科院分区：
文献类型：
--
作者：
Zhang MS;Brunner SF;Huguenin-Dezot N;Liang AD;Schmied WH;Rogerson DT;Chin JW
蛋白质中苏氨酸残基的磷酸化调控真核细胞中的各种过程，已鉴定了数千种苏氨酸磷酸化。苏氨酸磷酸化如何调节生物功能的理解将通过生物合成特定的磷酸蛋白的一般方法来加速。在这里，我们通过将平行的阳性选择与深度测序和统计分析相结合，解决了当前发现将非天然氨基酸结合到蛋白质中的氨基酰-tRNA合成酶/tRNA对的局限性，以创建一种直接发现选择性地结合非天然底物的氨基酰-tRNA合成酶/tRNA对的快速方法。我们的方法是可扩展的，并且能够直接发现具有相互正交的底物特异性的氨酰-tRNA合成酶/tRNA对。我们在细胞中生物合成了磷酸苏氨酸，并使用我们的新选择方法发现了一对磷酸苏氨酰-tRNA合成酶/tRNACUA对。通过结合这些进展，我们创建了一条将磷酸苏氨酸整合到蛋白质中的完全生物合成路线，并生物合成了几种磷蛋白；使磷蛋白结构确定和合成蛋白激酶激活成为可能。
The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to bio-synthesize defined phospho-proteins. Here we address limitations in current methods for discovering aminoacyl-tRNA synthetase/tRNA pairs for incorporating non-natural amino acids into proteins, by combining parallel positive selections with deep sequencing and statistical analysis, to create a rapid approach for directly discovering aminoacyl-tRNA synthetase/tRNA pairs that selectively incorporate non-natural substrates. Our approach is scalable and enables the direct discovery of aminoacyl-tRNA synthetase/tRNA pairs with mutually orthogonal substrate specificity. We biosynthesize phosphothreonine in cells, and use our new selection approach to discover a phosphothreonyl-tRNA synthetase/tRNACUA pair. By combining these advances we create an entirely biosynthetic route to incorporating phosphothreonine in proteins and biosynthesize several phosphoproteins; enabling phosphoprotein structure determination and synthetic protein kinase activation.