A genetically encoded fluorescent amino acid
A genetically encoded fluorescent amino acid
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DOI：
10.1073/pnas.0603965103
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发表时间：
2006-06-27
影响因子：
11.1
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
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作者：
Summerer, Daniel;Chen, Shuo;Schultz, Peter G.
将荧光团选择性引入蛋白质的能力为研究体外和体内蛋白质结构、动力学、定位和生物分子相互作用提供了强大的工具。在这里，我们报告了一种将低分子量荧光团选择性且有效地生物合成掺入蛋白质特定位点的策略。荧光氨基酸 2-氨基3-(5-(二甲氨基)萘-1-磺酰胺)丙酸（丹磺丙氨酸）是通过使用琥珀无义密码子和相应的正交 tRNA/氨酰基-tRNA 合成酶对在酿酒酵母中进行遗传编码的。这种环境敏感的荧光团被选择性地引入人超氧化物歧化酶中，并用于监测氯化胍存在下蛋白质的解折叠。这里描述的策略应该适用于原核和真核生物中的许多不同荧光团，并且它应该促进蛋白质结构和功能的生化和细胞研究。
The ability to introduce fluorophores selectively into proteins provides a powerful tool to study protein structure, dynamics, localization, and biomolecular interactions both in vitro and in vivo. Here, we report a strategy for the selective and efficient biosynthetic incorporation of a low-molecular-weight fluorophore into proteins at defined sites. The fluorescent amino acid 2-amino3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (dansylalanine) was genetically encoded in Saccharomyces cerevisiae by using an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. This environmentally sensitive fluorophore was selectively introduced into human superoxide dismutase and used to monitor unfolding of the protein in the presence of guanidinium chloride. The strategy described here should be applicable to a number of different fluorophores in both prokaryotic and eukaryotic organisms, and it should facilitate both biochemical and cellular studies of protein structure and function.