Research School of Chemistry , Australian National University , Canberra , ACT 2601 , Australia.
Monash Institute of Pharmaceutical Sciences , Monash University , Parkville , VIC 3052 , Australia.
Bioconjug Chem. 2018 Jul 18;29(7):2257-2264. doi: 10.1021/acs.bioconjchem.8b00254. Epub 2018 Jun 21.
Selenocysteine (Sec) is a naturally occurring amino acid that is also referred to as the 21st amino acid. Site-specific incorporation of Sec into proteins is attractive, because the reactivity of a selenol group exceeds that of a thiol group and thus allows site-specific protein modifications. It is incorporated into proteins by an unusual enzymatic mechanism which, in E. coli and other organisms, involves the recognition of a selenocysteine insertion sequence (SECIS) in the mRNA of the target protein. Reengineering of the natural machinery for Sec incorporation at arbitrary sites independent of SECIS elements, however, is challenging. Here we demonstrate an alternative route, whereby a photocaged selenocysteine (PSc) is incorporated as an unnatural amino acid in response to an amber stop codon, using a mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase, Mm PCC2RS, and its cognate tRNA. Following decaging by UV irradiation, proteins synthesized with PSc are readily tagged, e.g., with NMR probes to study ligand binding by NMR spectroscopy. The approach provides a facile route for genetically encoded Sec incorporation. It allows the production of pure selenoproteins and the Sec residue enables site-specific covalent protein modification with reagents that would usually react first with naturally occurring cysteine residues. The much greater reactivity of Sec residues allows their selective alkylation in the presence of highly solvent-exposed cysteine residues.
硒代半胱氨酸（Sec）是一种天然存在的氨基酸，也被称为第 21 种氨基酸。将 Sec 特异性地掺入蛋白质中很有吸引力，因为硒醇基团的反应性超过了硫醇基团，因此可以实现蛋白质的特异性修饰。它通过一种不寻常的酶促机制掺入蛋白质中，在大肠杆菌和其他生物体中，该机制涉及到靶蛋白 mRNA 中硒代半胱氨酸插入序列（SECIS）的识别。然而，独立于 SECIS 元件在任意位置对天然 Sec 掺入机制进行重新设计具有挑战性。在这里，我们展示了一种替代途径，即使用突变的 Methanosarcina mazei 吡咯赖氨酸-tRNA 合成酶 Mm PCC2RS 及其同源 tRNA，通过光笼硒代半胱氨酸（PSc）作为非天然氨基酸掺入，响应琥珀终止密码子。用紫外线照射去笼后，用 PSc 合成的蛋白质很容易被标记，例如用 NMR 探针通过 NMR 光谱研究配体结合。该方法为基因编码的 Sec 掺入提供了一种简便的途径。它允许生产纯硒蛋白，并且 Sec 残基允许用试剂进行特异性的共价蛋白质修饰，这些试剂通常首先与天然半胱氨酸残基反应。Sec 残基的反应性要强得多，因此可以在高度暴露于溶剂的半胱氨酸残基存在下选择性地对其进行烷基化。
