Heim R, Prasher D C, Tsien R Y
Howard Hughes Medical Institute, University of California, San Diego, La Jolla 92093-0647.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12501-4. doi: 10.1073/pnas.91.26.12501.
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
维多利亚多管发光水母的绿色荧光蛋白（GFP）是一种不同寻常的蛋白质，它具有强烈的可见光吸收能力，其发色团为对羟基苄叉咪唑啉酮，能发出荧光，该发色团是由蛋白质自身65 - 67位的丝氨酸 - 酪氨酸 - 甘氨酸序列环化和氧化产生的。cDNA的克隆以及荧光蛋白在多种生物体中的异源表达表明，这种独特的翻译后修饰必定是自发的，或者仅依赖于普遍存在的酶和反应物。我们报告称，最终荧光团的形成需要分子氧，且其进行过程具有一个与稀释无关的时间常数（在22℃和大气氧分压下约为4小时），这意味着氧化过程不需要酶或辅因子。对GFP进行诱变并筛选光谱改变的变体。最引人注目的突变体发出蓝色荧光，并且在66位酪氨酸处含有组氨酸。两种明显不同颜色的可得性应能通过实现体内差异基因表达和蛋白质定位的可视化以及通过荧光共振能量转移测量蛋白质结合，显著扩展GFP在分子和细胞生物学中的用途。
