Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Nat Chem. 2016 Nov;8(11):1027-1034. doi: 10.1038/nchem.2573. Epub 2016 Jul 25.
Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).
用小分子在活细胞中完全特异性地控制蛋白质的功能是非常理想的，但具有挑战性。在这里，我们报告了一种小分子开关，可以用来控制蛋白质活性。该方法利用膦介导的施蒂丁格还原来激活蛋白质功能。通过使用吡咯赖氨酸 tRNA 合成酶/tRNA 对在哺乳动物细胞中对邻叠氮苯甲氧基羰基氨基酸进行遗传编码，能够在感兴趣的蛋白质中特异性地引入可去除小分子的保护基团。通过生物正交的施蒂丁格还原将该基团脱保护，从而将活性野生型蛋白质递送到蛋白质中，从而使蛋白质失活。该方法已应用于几种细胞过程的条件控制，包括生物发光（荧光素酶）、荧光（增强型绿色荧光蛋白）、蛋白质易位（核定位序列）、DNA 重组（Cre）和基因编辑（Cas9）。
