Department of Chemistry, Binghamton University, State University of New York, 13902, USA.
ACS Chem Biol. 2010 Aug 20;5(8):777-85. doi: 10.1021/cb100060v.
A fluorescent probe has been attached to the carboxy terminus of the alpha-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.
通过酶反应和化学反应，将荧光探针连接到α，β-微管蛋白的α亚基的羧基末端。通过使用微管蛋白酪氨酸连接酶，将非天然氨基酸 3-甲酰基酪氨酸连接到微管蛋白的羧基末端。非天然氨基酸的芳醛作为正交亲电试剂，特异性地与含有芳族腙官能团的荧光团反应，在这种情况下，该荧光团为 7-肼基-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温和，使荧光标记的微管蛋白保持其组装成微管的能力。标记反应的一个关键特征是，它会导致荧光团的吸收和发射最大值发生红移，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体外系统。
