Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein
Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein
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DOI：
10.1016/s0969-2126(98)00127-0
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发表时间：
1998-10-01
期刊：
影响因子：
5.7
通讯作者：
Remington, SJ
中科院分区：
文献类型：
--
作者：
Wachter, RM;Elsliger, MA;Remington, SJ
背景资料：由于其自发产生自身荧光团的能力，来自水母Aequorea维多利亚的绿色荧光蛋白（GFP）被广泛用作分子和细胞生物学中的荧光标记物。黄色荧光蛋白（YFP）具有迄今为止检查的所有GFP变体的最长波长发射。这种光谱的变化是T203 Y取代（单字母氨基酸代码）的结果，T203 Y取代是基于GFP S65 T的X射线结构合理设计的突变。结果：我们已经确定了YFP T203 Y/S65 G/V68 L/S72 A和YFP H148 G的晶体结构，分辨率分别为2.5和2.6埃。两种结构均显示Tyr 203和发色团之间几乎共面的π-π堆叠的清晰电子密度。与其他可用的结构相比，发色团已被置换近1 A。尽管H148 G突变导致产生了通向发色团腔的溶剂通道，但仍保持了强烈的荧光。可以滴定完整蛋白质中的发色团，并且两种变体具有7.0（YFP）和8.0（YFP H148 G）的pK（a）值。结论：T203 Y YFP变体的观察到的红移被提出主要是由于π堆叠的Tyr 203的额外极化。发色团位置的改变表明，附近残基的确切位置对于发色团形成的化学过程并不重要。YFP显著扩展了GFP可用作活细胞中的pH指示剂的pH范围。
Background: Because of its ability to spontaneously generate its own fluorophore, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria is used extensively as a fluorescent marker in molecular and cell biology. The yellow fluorescent proteins (YFPs) have the longest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-letter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T.Results: We have determined the crystal structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 Angstrom resolution, respectively. Both structures show clear electron density for nearly coplanar pi-pi stacking between Tyr203 and the chromophore. The chromophore has been displaced by nearly 1 A in comparison to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, intense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pK(a) values of 7.0 (YFP) and 8.0 (YFP H148G).Conclusions: The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.