EAPSI: Monitoring High Resolution Broadband Fluorescence of the Green Fluorescent Protein Model Chromophore

绿色荧光蛋白(GFP)的发现定义了可视化细胞事件和生命过程的方式。通过在超分辨率显微镜中使用GFP作为一种可遗传编码的荧光试剂，非侵入性活细胞成像推动了生命科学的众多进步。对于GFP发色团在蛋白质口袋中是如何工作的，以及为什么它的荧光在溶液中消失，目前还不清楚。这一认识差距阻碍了我们对导致有效排放的重要因素的理解。新西兰惠灵顿维多利亚大学将与新开发的瞬变光栅光致发光光谱(TGPLS)专家贾斯汀·霍奇基斯博士合作进行研究。这一独特的机会将使我们能够在固有的分子时间尺度上研究GFP生色团在溶液中的宽带荧光信号，并将进一步有助于表征蛋白质中生色团的荧光状态。为了设计用于高分辨率显微镜的改进的荧光蛋白质，基本了解动态GFP生色团周围的氢键网络是至关重要的。在蛋白质内部，锁定的丝氨酸-酪氨酸-甘氨酸发色团不能异构化，不可避免地被迫通过荧光释放能量。在蛋白质基质之外，荧光和异构化之间的相互作用使量子产率从~0.8下降到~2×10-4。这种显著的荧光变化表明，4-hydroxybenzylidene-1，2-dimethylimidazolinone(HBDi)的激发态布居通过从S1到S0的锥形交点，而其余的则通过荧光进行。由于具有空间和时间分辨率，它是研究HBDI的时间分辨荧光分布和进一步了解光激发后的电子激发态中间体的重要工具。与霍奇基斯博士的合作将有助于描绘荧光是如何从HBDI激发态的一小部分产生的，并使我们能够更好地了解蛋白质基质在荧光中所起的作用。该奖项由东亚和太平洋夏季研究所计划设立，支持一名美国研究生的夏季研究，由NSF和新西兰皇家学会联合资助。

The discovery of the Green Fluorescent Protein (GFP) has defined the way to visualize cellular events and life processes. By using GFP as a genetically encodable fluorescence reagent in super-resolution microscopy, non-invasive live cell imaging has powered numerous advances in life sciences. A mechanistic understanding of how the GFP chromophore works in the protein pocket, and why its fluorescence vanishes in solution, remains unclear. This knowledge gap hinders our understanding of important factors which lead to efficient emission. In collaboration with Dr. Justin Hodgkiss, an expert on the newly developed Transient Grating Photoluminescence Spectroscopy (TGPLS), research will be conducted at Victoria University of Wellington, New Zealand. This unique opportunity will allow the study of the broadband fluorescence signal of the GFP chromophore in solution on the intrinsic molecular timescale and will further help to characterize the fluorescent state of the chromophore within the protein.To design improved fluorescent proteins for high-resolution microscopy, a fundamental understanding of the hydrogen bonding network around a dynamic GFP chromophore is critical. Within the protein, the locked Ser-Tyr-Gly chromophore cannot isomerize and is inevitably forced to release energy through fluorescence. Outside the protein matrix, the interplay between fluorescence and isomerization drops the quantum yield from ~0.8 to ~2x10-4. This striking change of fluorescence indicates that the excited state population of 4-hydroxybenzylidene-1,2-dimethylimidazolinone (HBDI) travels through a conical intersection from S1 to S0, while the rest proceeds through fluorescence. Because of both the spatial and temporal resolution of TGPLS, it is a prime tool to study the time-resolved fluorescence profile of HBDI and gain further insights into the electronic excited state intermediates after photoexcitation. Collaboration with Dr. Hodgkiss will help delineate how fluorescence occurs from a small portion of the HBDI excited state and allow us to better understand the role the protein matrix plays in fluorescence.This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the Royal Society of New Zealand.