Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors
Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors
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DOI：
10.1016/j.str.2017.12.006
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发表时间：
2018-02-06
期刊：
影响因子：
5.7
通讯作者：
Liu, Ce Feng
中科院分区：
文献类型：
--
作者：
Campbell, Benjamin C.;Petsko, Gregory A.;Liu, Ce Feng
我们测定了最亮的荧光蛋白之一三叶草的晶体结构，发现其相对于野生型GFP的T203H/S65G突变将关键的E222侧链锁定在一个固定的构型中，该构象模仿了EGFP中的主要构象。由此产生的平衡漂移到主要是去质子化的发色团增加了消光系数(EC)，反对光激活，并对变色漂移负责。三叶草的亮度还可以归因于H203和发色团之间的pi-pi堆积作用。与这些观察结果一致的是，三叶草G65S突变体逆转了平衡位移，显著降低了EC，并使三叶草在激活可光激活GFP的条件下可光激活。利用三叶草结构，我们合理地设计了非光活化氧化还原传感器roClover1，并确定了它的结构及其亲本模板roClover0.1的结构。这些高分辨率结构提供了对GFP结构-功能关系的更深层次的了解，并可能有助于开发激发改进型比率生物传感器。
We have determined the crystal structure of Clover, one of the brightest fluorescent proteins, and found that its T203H/S65G mutations relative to wild-type GFP lock the critical E222 side chain in a fixed configuration that mimics the major conformer of that in EGFP. The resulting equilibrium shift to the predominantly deprotonated chromophore increases the extinction coefficient (EC), opposes photoactivation, and is responsible for the bathochromic shift. Clover's brightness can further be attributed to a pi-pi stacking interaction between H203 and the chromophore. Consistent with these observations, the Clover G65S mutant reversed the equilibrium shift, dramatically decreased the EC, and made Clover photoactivatable under conditions that activated photoactivatable GFP. Using the Clover structure, we rationally engineered a non-photoactivatable redox sensor, roClover1, and determined its structure as well as that of its parental template, roClover0.1. These high-resolution structures provide deeper insights into structure-function relationships in GFPs and may aid the development of excitation-improved ratiometric biosensors.