Site-Specific Orthogonal Labeling of the Carboxy Terminus of α-Tubulin
Site-Specific Orthogonal Labeling of the Carboxy Terminus of α-Tubulin
复制标题
DOI：
10.1021/cb100060v
复制
发表时间：
2010-08-01
影响因子：
4
通讯作者：
Bane, Susan
中科院分区：
文献类型：
--
作者：
Banerjee, Abhijit;Panosian, Timothy D.;Bane, Susan
通过酶反应和化学反应，将荧光探针连接到α，β-微管蛋白α，β-微管蛋白α-亚基的羧基末端。非天然氨基酸3-甲酰酪氨酸通过微管蛋白酪氨酸连接酶连接到α-微管蛋白的羧基末端。非天然氨基酸的芳香醛用作与含有芳香肼官能团的荧光团特异反应的正交电泳体，在这种情况下，该荧光团是7-肼-4-甲基香豆素。非天然氨基酸和荧光团之间形成共价键的条件很温和，允许荧光标记的微管蛋白保持其组装成微管的能力。该标记反应的一个关键特征是它使荧光团的吸收和发射峰值发生红移，并伴随着其量子产率的增加；因此，在未反应的荧光团存在的情况下，可以观察到荧光标记的蛋白质。酶反应和偶联反应都可以在活细胞中发生。这里提出的方法应该适用于各种各样的体外系统。
A fluorescent probe has been attached to the carboxy terminus of the a-subunit of alpha,beta-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of alpha-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore's absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.