Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
J Am Chem Soc. 2010 Mar 31;132(12):4086-8. doi: 10.1021/ja910688s.
Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.
通过在特定位置引入笼状基团，可以实现对蛋白质功能的精确光化学控制。已经使用化学和酶方法（包括体外翻译和化学连接）在体外对蛋白质进行光笼化。这些方法已经扩展到允许通过透化或显微注射将光笼蛋白引入细胞，但细胞递送仍然具有挑战性。由于赖氨酸残基是核定位序列的关键决定因素、关键翻译后修饰（包括泛素化、甲基化和乙酰化）的靶标以及许多重要酶活性位点的关键残基，我们对光笼化赖氨酸以控制蛋白质定位、翻译后修饰和酶活性感兴趣。以前在活细胞中尚未证明通过蛋白质中的赖氨酸残基对这些重要功能进行光化学控制。在这里，我们合成了 1 并进化了一个吡咯赖氨酸-tRNA 合成酶/tRNA 对，以在哺乳动物细胞中响应琥珀终止密码子遗传编码该氨基酸的掺入。为了说明这种氨基酸的用途，我们将核定位序列（NLS）的核蛋白和肿瘤抑制因子 p53 笼化在人细胞中，从而将蛋白质错误定位在细胞质中。我们用光脉冲触发蛋白质核输入，从而使我们能够直接定量核输入的动力学。
