研究绿色荧光蛋白氧化光转化的分子机制

Photoinduced reactions play an important role in the photocycle of fluorescent proteins family. The chromophore jumps into the excited state from the ground state by absorbing a photon. The different relaxation channels lead to the different optical properties of fluorescent proteins. And fluorescence is the main relaxation channel restoring the ground-state chromophore. Light can be used to selectively activate or deactivate fluorescent proteins. In some fluorescent proteins this can permit photoconversion , which is the fluorescence switches between two colors (e.g., from green to red). For a long time, photoconversions were perceived as an unusual property of a few outliers from the large fluorescent protein family. The ability to undergo photoconversions was attributed to a specific amino-acid environment conductive of intramolecular reactions involving the chromophore and leading to its chemical modification. This paradigm substantially shifted in 2009, when several new photoconversions were described. One of them is the so-called photooxidative redding (green to red photoconversion in the presence of oxidants), which occurs in a variety of fluorescent proteins that have anionic eGFP-like chromphore. Subsequent studies provided additional examples of the ubiquity of photoconversion phenomena. No structural information about the red chromophore in eGFP-like proteins is available, although several hypotheses were put forward. In this proposal, we are aiming to determinating the crystal structure of the red form by the X-ray crystallography. Based on the results, we then design some mutants, and monitor the molecular reaction process by the ultrafast spectroscopy. Finally, a possible molecular interaction mechanism is proposed. In addition, this study is not only helpful to understand the biological functions of GFP-like protein family, but also plays an important role to choose and design reasonable fluorescent proteins.

荧光蛋白的发光现象与光反应密不可分。生色团吸收光子后由基态跃迁到激发态，而激发态的弛豫方式决定了荧光蛋白不同的光学性质，其中发射荧光是最主要的弛豫方式。光转化现象是荧光蛋白在两种颜色状态间转换，人们一直认为只有少数特殊的荧光蛋白才有的这种性质。直到2009年发现含有阴离子GFP-like生色团的荧光蛋白，在氧化剂存在下荧光颜色也会由绿色变为红色。目前对这一现象只提出了两种假设机理模型，并没有相关的晶体结构信息。本项目拟通过得到氧化剂存在下的红色荧光的晶体，用X射线晶体学方法收集衍射数据并解析结构，应用定点突变构建突变体；并用超快光谱学确定生色团的每个结构变化所需要的时间历程和发生的先后顺序。结合X射线晶体学和超快光谱学结果，提出一个光转化分子机理。这一机理的揭示不仅能帮助人们理解荧光蛋白家族的本质功能，对人们设计和选择荧光蛋白也有重要的指导意义。