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

职业：利用飞秒受激拉曼光谱揭示新兴荧光蛋白生物传感器的荧光机制

• 批准号: 1455353
• 负责人: Chong Fang
• 金额: $ 65万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-15 至 2021-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1455353&HistoricalAwards=false
• 关键词: CAREER; Revealing; fluorescence; mechanisms; emerging

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.

凭借这一职业奖，化学部的生命过程化学项目和化学结构与动力学A项目正在资助俄勒冈州立大学的Chong Fang博士研究新兴荧光蛋白生物传感器的荧光机制。这些色彩缤纷的生物分子最初源自水母，后来又源自珊瑚礁，近二十年来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光稳定性、亮度、穿透深度和颜色对比度有限等缺点，阻碍了它们在物理和生命科学中的进一步应用。改进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，其中涉及蛋白质口袋中心的高度动态的发色团。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。用于荧光蛋白研究的新飞秒拉曼光谱方法，以及随后影响年轻人思想的课程开发和外展研讨会，将产生分子“电影”，展示嵌入的发色团如何响应光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 学习者创造互动和可持续的参与文化。该研究的科学重点是利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，阐明控制与绿色荧光蛋白 (GFP) 相关的蛋白质和生物传感器的荧光特性的发色团运动。光激发发色团的结构演化将通过从零时间开始的瞬态振动峰进行监测，通过动力学分析进行量化，产生关键的时间常数，并伴随计算化学来绘制原子轨迹。将研究沿多维反应坐标的低频骨骼运动的功能相关性，特别是在亚皮秒时间尺度上。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；新实验将使对新兴 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进行系统评估成为可能。这项研究的见解将丰富物理化学和生物物理学，并有可能指导具有改进性能的下一代 FP 生物传感器的合理设计。

项目成果

Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission

观察工程钙生物传感器的发光：发射前反应途径的改变

• DOI: 10.1021/acs.jpcb.8b10587
• 发表时间: 2018
• 期刊: The Journal of Physical Chemistry B
• 作者: Tachibana, Sean R.;Tang, Longteng;Zhu, Liangdong;Liu, Weimin;Wang, Yanli;Fang, Chong
• 通讯作者: Fang, Chong

Discovering a rotational barrier within a charge-transfer state of a photoexcited chromophore in solution

• DOI: 10.1063/1.5143441
• 发表时间: 2020-03-01
• 期刊: STRUCTURAL DYNAMICS-US
• 影响因子: 2.8
• 作者: Krueger, Taylor D.;Boulanger, Sean A.;Fang, Chong
• 通讯作者: Fang, Chong

Sum-Frequency-Generation-Based Laser Sidebands for Tunable Femtosecond Raman Spectroscopy in the Ultraviolet

• DOI: 10.3390/app5020048
• 发表时间: 2015-04
• 期刊: Applied Sciences
• 作者: Liangdong Zhu;Weimin Liu;Yanli Wang;Chong Fang

Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores

• DOI: 10.3389/fmolb.2020.00131
• 发表时间: 2020-07-07
• 期刊: FRONTIERS IN MOLECULAR BIOSCIENCES
• 影响因子: 5
• 作者: Oscar, Breland G.;Zhu, Liangdong;Fang, Chong
• 通讯作者: Fang, Chong

Ultrafast excited-state proton transfer dynamics in dihalogenated non-fluorescent and fluorescent GFP chromophores

• DOI: 10.1063/1.5138666
• 发表时间: 2020-01-14
• 期刊: JOURNAL OF CHEMICAL PHYSICS
• 影响因子: 4.4
• 作者: Chen, Cheng;Zhu, Liangdong;Fang, Chong
• 通讯作者: Fang, Chong