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作为基因可编码的荧光试剂，非侵入性活细胞成像为生命科学的许多进步提供了动力。关于GFP发色团如何在蛋白质口袋中起作用的机制理解，以及为什么它的荧光在溶液中消失，仍然不清楚。这种知识差距阻碍了我们对导致高效排放的重要因素的理解。与新开发的瞬态光栅光致发光光谱（TGPLS）专家Justin Hodgkiss博士合作，研究将在新西兰惠灵顿维多利亚大学进行。这一独特的机会将允许在固有分子时间尺度上研究溶液中GFP发色团的宽带荧光信号，并将进一步有助于表征蛋白质内发色团的荧光状态。为了设计用于高分辨率显微镜的改进荧光蛋白，对动态GFP发色团周围的氢键网络的基本理解是至关重要的。在蛋白质内部，锁定的ser - tyrr - gly发色团不能异构化，不可避免地被迫通过荧光释放能量。在蛋白质基质外，荧光和异构化之间的相互作用使量子产率从~0.8降至~2x10-4。这种显著的荧光变化表明，4-羟基苄基-1,2-二甲基咪唑啉酮（HBDI）的激发态居群从S1到S0经过一个锥形相交，而其余居群则通过荧光进行。由于TGPLS具有空间和时间分辨率，因此它是研究HBDI时间分辨荧光谱和进一步了解光激发后电子激发态中间体的主要工具。与Hodgkiss博士的合作将有助于描述荧光如何从HBDI激发态的一小部分发生，并使我们更好地理解蛋白质基质在荧光中所起的作用。该奖项由美国国家科学基金会和新西兰皇家学会共同资助，隶属于东亚和太平洋暑期学院项目，支持一名美国研究生进行暑期研究。

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.