Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein
Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein
复制标题
绘制为大斯托克斯位移红色荧光蛋白提供动力的完整光循环
DOI：
10.1002/anie.202212209
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Weimin Liu
中科院分区：
文献类型：
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作者：
Ziyu Wang;Ya Zhang;Cheng Chen;Ruixue Zhu;Jiaming Jiang;Tsu‐Chien Weng;Quanjiang Ji;Yifan Huang;Chong Fang;Weimin Liu
大斯托克斯位移 (LSS) 红色荧光蛋白 (RFP) 对于生物成像的进步非常理想。 RFP mKeima 具有共存的顺式和反式异构体，作为激发态质子转移 (ESPT) 引起的 LSS 发射的原型系统具有重要意义，但其机制仍然难以捉摸。我们在量子计算的辅助下实施了飞秒受激拉曼光谱（FSRS）和各种时间分辨电子光谱，以剖析从 ESPT、异构化到溶液中基态质子转移的顺式和反式 mKeima 光循环。这项工作体现了 FSRS 通过全局分析来解析中间态拉曼指纹的能力。重要的是，去质子化反式异构体控制 620nm 处的 LSS 发射，而去质子化顺式异构体的 520nm 发射由于超快的顺式转反式异构化而较弱。因此，补充光谱技术作为桌面工具集对于研究生理环境中的光化学至关重要。
Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexistingcis‐andtrans‐isomers, holds significance as an archetypal system for LSS emission due to excited‐state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time‐resolved electronic spectroscopies, aided by quantum calculations, to dissect thecis‐ andtrans‐mKeima photocycle from ESPT, isomerization, to ground‐state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonatedtrans‐isomer governs LSS emission at 620 nm, while the deprotonatedcis‐isomer's 520 nm emission is weak due to an ultrafastcis‐to‐transisomerization. Complementary spectroscopic techniques as a table‐top toolset are thus essential to study photochemistry in physiological environments.