Crystal Structure of a New Cyan Fluorescent Protein and Its Hue-Shifted Variants
Crystal Structure of a New Cyan Fluorescent Protein and Its Hue-Shifted Variants
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DOI：
10.1021/bi801658p
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发表时间：
2009-06-16
期刊：
影响因子：
2.9
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
--
作者：
Kikuchi, Akihiro;Fukumura, Eiko;Miyawaki, Atsushi
基于绿色荧光蛋白（GFP）的技术在分子生物学中已经很成熟；然而，对荧光颜色进行微调的详细机制仍不清楚。在此，我们报道了一种新的发射青色光的类GFP蛋白KCy的克隆及晶体结构。我们还开发了一种具有高折叠效率的突变蛋白（KCy - G4219：吸收波长λ(abs)=453nm；发射波长λ(em)=486nm）。X射线衍射分析表明，KCy的发色团由内部的丝氨酸62 - 酪氨酸63 - 甘氨酸64三肽形成。形成发色团的三肽中第一位的丝氨酸残基具有短的极性链（-OH），它与组氨酸38的咪唑在2.96埃的距离处形成非共价相互作用。在KCy - G4219中，将组氨酸38替换为谷氨酰胺（KCy - R1）或亮氨酸残基，分别导致发射峰最大值从486nm略微但显著地红移至492nm或496nm。分辨率为1.58埃的KCy - R1的晶体结构表明，丝氨酸62 - OH与替换后的谷氨酰胺38之间的非共价相互作用的距离（3.07埃）比在野生型KCy中观察到的更长。这种相互作用在亮氨酸突变体中不存在，这表明这种相互作用是负责微调发射峰最大值的关键因素之一，发射峰最大值受发色团极化的影响。此外，结构比较表明，埋在丙氨酸158残基和发色团酚盐之间空间的一个额外水分子也对发色团极化有影响。
Green fluorescent protein (GFP) based techniques are well established in molecular biology; however, the detailed mechanism for the fine-tuning of fluorescent colors remains unclear. Here, we report the cloning and crystal structure of a new cyan-emitting GFP-like protein, KCy. We also developed a mutant protein with a high folding efficiency (KCy-G4219: lambda(abs) = 453 nm; lambda(em) = 486 nm). X-ray diffraction analysis revealed that the KCy chromophore is formed from an internal Ser62-Tyr63-Gly64 tripeptide. The serine residue at the first position of the chromophore-forming tripeptide has a short polar chain (-OH) that forms a noncovalent interaction with the His38 imidazole at a distance of 2.96 angstrom. Substitution of His38 in KCy-G4219 with Gln (KCy-R1) or Lieu residues resulted in a slight but significant red shift of the emission peak maximum from 486 to 492 or 496 rim, respectively. The crystal structure of KCy-R1 deter-mined at a resolution of 1.58 angstrom showed that the noncovalent interaction between Ser62-OH and the substituted Gln38 occurred over a longer distance (3.07 angstrom) than that observed in the wild-type KCy. Such an interaction is absent in the Leu mutant, suggesting that this interaction is one of the key factors responsible for fine-tuning the emission peak maxima, which are affected by chromophore polarization. Moreover, the structural comparison suggests that an additional water molecule buried in the space between the Ala 158 residue and the chromophore phenolate is also responsible for the chromophore polarization.