Collaborative Research: Dissecting photoconversion in fluorescent proteins frame by frame

合作研究：逐帧剖析荧光蛋白中的光转换

• 批准号: 1817949
• 负责人: Chong Fang
• 金额: $ 40万
• 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: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817949&HistoricalAwards=false
• 关键词: Collaborative; Research; Dissecting; photoconversion; fluorescent

This project will investigate the mechanism of irreversible color conversion, as observed in a set of fluorescent proteins derived from jellyfish and reef corals. This group of proteins, termed green-to-red photoconvertible fluorescent proteins (pcFPs), has greatly advanced molecular and cellular imaging techniques in the life sciences. Notably, the light-induced change in appearance has played a critical role in the development of super-resolution methods to image subcellular events beyond the diffraction limit of visible light. The objective of this project is to unravel the connection between protein motion and the rearrangement of chemical bonds, an interplay that is ultimately responsible for the development of red color. Ultrafast spectroscopic methods and computer modeling will be combined with time-dependent biochemical measurements on pcFPs. The correlation of dynamical and chemical processes will help elucidate novel principles in photobiology and enzymology. This work will facilitate the training of undergraduate and graduate students, as well as the training of post-doctoral researchers. In addition, this project will serve as a cornerstone for a number of outreach activities. To enhance evidence-based instructional practices, cutting-edge research on pcFPs will be integrated into formal coursework. The overarching goal of this work is to investigate how ultrafast atomic motions in a protein's active site are linked to long-range dynamics and catalytic functionality. To develop a mechanistic model that connects protein motions to key chemical events, femtosecond stimulated Raman spectroscopy (FSRS) will be combined with photoconversion kinetic measurements and molecular dynamics simulations. Currently, wavelength-tunable FSRS is the only table-top technique that can track bond vibrations starting with photoexcitation. Mechanistic hypotheses will be tested by evaluating rate and equilibrium constants of pcFPs engineered to bear modified building blocks. With this synergistic approach, functional models will be developed that integrate ultrafast atomic motions, local side chain rotations and global chain dynamics on the pathway of color modification.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

这个项目将研究从水母和珊瑚礁中提取的一组荧光蛋白中观察到的不可逆转颜色转换的机制。这组蛋白质被称为绿色到红色的光可转换荧光蛋白(PCFP)，在生命科学中具有非常先进的分子和细胞成像技术。值得注意的是，光诱导的外观变化在超分辨率方法的发展中发挥了关键作用，以成像超出可见光衍射极限的亚细胞事件。这个项目的目标是揭示蛋白质运动和化学键重排之间的联系，化学键的重排是最终导致红色发展的一种相互作用。超快光谱方法和计算机模拟将与PCFP上依赖时间的生化测量相结合。动力学和化学过程的关联将有助于阐明光生物学和酶学中的新原理。这项工作将为本科生和研究生的培养以及博士后研究人员的培养提供便利。此外，该项目将作为若干外联活动的基石。为了加强循证教学实践，将把关于PCFP的尖端研究纳入正式课程。这项工作的首要目标是调查蛋白质活性部位的超快原子运动是如何与远程动力学和催化功能联系起来的。为了开发一个将蛋白质运动与关键化学事件联系起来的机制模型，飞秒受激拉曼光谱(FSRs)将与光转化动力学测量和分子动力学模拟相结合。目前，波长可调的FSR是唯一一种可以从光激发开始跟踪键振动的桌面技术。机械假说将通过评估PCFP的速率和平衡常数来检验，这些PCFP被设计成承载修改的构建块。通过这种协同方法，将开发出功能模型，将超快原子运动、局部侧链旋转和全球链动力学整合到颜色修饰的路径上。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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

Ultrafast Dynamics and Photoresponse of a Fungi‐Derived Pigment Xylindein from Solution to Thin Films

真菌衍生色素 Xylindein 从溶液到薄膜的超快动力学和光响应

• DOI: 10.1002/chem.202005155
• 发表时间: 2021
• 期刊: Chemistry – A European Journal
• 作者: Krueger, Taylor D.;Giesbers, Gregory;Van Court, Ray C.;Zhu, Liangdong;Kim, Ryan;Beaudry, Christopher M.;Robinson, Seri C.;Ostroverkhova, Oksana;Fang, Chong
• 通讯作者: Fang, Chong

Photoswitchable Fluorescent Proteins: Mechanisms on Ultrafast Timescales

• DOI: 10.3390/ijms23126459
• 发表时间: 2022-06-09
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Tang, Longteng;Fang, Chong
• 通讯作者: Fang, Chong

Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein

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
• 发表时间: 2022
• 期刊: Angewandte Chemie International Edition
• 作者: Ziyu Wang;Ya Zhang;Cheng Chen;Ruixue Zhu;Jiaming Jiang;Tsu‐Chien Weng;Quanjiang Ji;Yifan Huang;Chong Fang;Weimin Liu
• 通讯作者: Weimin Liu

Role of Hydroxyl Groups in the Photophysics, Photostability, and (Opto)electronic Properties of the Fungi-Derived Pigment Xylindein

• DOI: 10.1021/acs.jpcc.0c09627
• 发表时间: 2021-03-18
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY C
• 影响因子: 3.7
• 作者: Giesbers, Gregory;Krueger, Taylor D.;Ostroverkhova, Oksana
• 通讯作者: Ostroverkhova, Oksana