Luo Ji, Torres-Kolbus Jessica, Liu Jihe, Deiters Alexander
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania, 15260, USA.
Chembiochem. 2017 Jul 18;18(14):1442-1447. doi: 10.1002/cbic.201700147. Epub 2017 Jun 13.
We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl-tRNA synthetase/tRNA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off-to-on light switching. This reporter was then used to evaluate the activation rates of the different light-removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox.
我们通过在细菌和哺乳动物细胞中使用工程化的吡咯赖氨酰 - tRNA合成酶/tRNA对,对三种具有改善光化学性质的新型笼蔽酪氨酸类似物进行了基因编码。我们将新型酪氨酸类似物应用于通过笼蔽萤火虫荧光素酶的关键酪氨酸残基Tyr340来进行光调节,并观察到了出色的关闭到开启的光切换。然后使用该报告基因来评估活细胞中不同光可去除保护基团的活化速率。我们确定硝基亚胡椒基笼蔽基团是掺入效率和光活化性质之间的极佳折衷方案。为了证明新型笼蔽酪氨酸的适用性,我们对一种重要的蛋白酶——烟草蚀纹病毒(TEV)蛋白酶进行了光学控制工程改造。能够在活细胞中将不同的笼蔽酪氨酸类似物掺入蛋白质中,进一步扩展了非天然氨基酸和光遗传学工具箱。