CAREER: Revealing fluorescence mechanisms of emerging fluorescent protein biosensors using femtosecond stimulated Raman spectroscopy

有了这个CAREER奖，生命过程的化学计划和化学结构和动力学-化学部的一个计划正在资助来自俄勒冈州州立大学的方冲博士研究新兴荧光蛋白生物传感器的荧光机制。这些五颜六色的生物分子最初来源于水母，后来又来源于珊瑚礁，近二十年来，它们彻底改变了细胞成像和分子生物学。然而，这些生物传感器仍然存在诸如有限的光稳定性、亮度、穿透深度和颜色对比度等缺点，这阻碍了它们在物理和生命科学中的进一步应用。提高其功能的瓶颈是关于荧光蛋白的潜在结构动力学的信息非常有限，这涉及蛋白质口袋中心的高度动态发色团。这一追求将为研究生和本科生以及博士后学者提供获得超快光谱学专业培训的机会，特别是在分子振动领域和生物物理化学领域。新的飞秒拉曼光谱方法用于荧光蛋白的研究，随后是课程开发和外展研讨会，以影响年轻人的思想，将产生分子“电影”，展示嵌入的生色团如何对光作出反应，并编排单个质子的离开或芳香环的摆动。该项目还将提供综合教育计划，为STEM学习者创造一种互动和可持续的参与文化。该研究的科学重点是阐明控制绿色荧光蛋白（GFP）相关蛋白质和生物传感器的荧光特性的发色团运动，使用新开发的高分辨率和广泛可调的飞秒受激拉曼光谱技术。光激发发色团的结构演化将通过从时间零点开始的瞬态振动峰来监测，通过动力学分析来量化，从而产生关键的时间常数，并伴随着计算化学来绘制原子轨迹。低频骨骼运动的功能相关性沿着多维反应坐标，特别是在亚皮秒的时间尺度上，将进行研究。目前已发现野生型GFP中的酚环摆动运动可门控激发态质子转移;新实验将使系统评估新兴GFP相关生物传感器中发色团构象动力学和发射结果的控制因素成为可能。这项研究的见解将丰富物理化学和生物物理学，并有可能指导下一代FP生物传感器的合理设计。

With this CAREER award, the Chemistry of Life Processes Program and the Chemical Structure and Dynamics-A Program in the Chemistry Division are funding Dr. Chong Fang from Oregon State University to study the fluorescence mechanisms of emerging fluorescent protein biosensors. These colorful biomolecules originally derived from jellyfish and later from reef corals have revolutionized cellular imaging and molecular biology for almost two decades. However, these biosensors still suffer from drawbacks such as limited photostability, brightness, penetration depth, and color contrast, which hinder their further applications in physical and life sciences. The bottleneck in improving their functionality is the very limited information available about the underlying structural dynamics of fluorescent proteins, which involve a highly dynamic chromophore in the center of the protein pocket. This pursuit will provide graduate and undergraduate students as well as postdoctoral scholars the opportunity to acquire specialized training in ultrafast spectroscopy, particularly in the molecular vibrational domain, and in biophysical chemistry. The new femtosecond Raman spectroscopic method for fluorescent protein studies, followed by curriculum development and outreach workshops to impact young minds, will yield molecular "movies" showing how the embedded chromophore responds to light and choreographs the departure of a single proton or the swing of an aromatic ring. This project will also provide integrated educational programs to create an interactive and sustainable culture of engagement for STEM learners.The scientific focus of the research is to elucidate the chromophore motions that control the fluorescence characteristics of proteins related to Green Fluorescent Proteins (GFPs) and of biosensors using newly developed, high resolution, and broadly tunable femtosecond stimulated Raman spectroscopic techniques. The structural evolution of the photoexcited chromophore will be monitored by transient vibrational peaks starting from time zero, quantified by kinetic analysis yielding crucial time constants and accompanied by computational chemistry to map atomic trajectories. The functional relevance of low-frequency skeletal motions along the multidimensional reaction coordinate, especially on the subpicosecond timescale, will be studied. Currently a phenolic ring wagging motion in wild-type GFP has been found to gate excited state proton transfer; the new experiments will make possible the systematic evaluation of governing factors for the chromophore conformational dynamics and emission outcomes in emerging GFP-related biosensors. Insights from this study will enrich physical chemistry and biophysics, and have the potential to guide the rational design of next-generation FP-biosensors with improved properties.