Collaborative Research: Dissecting photoconversion in fluorescent proteins frame by frame

本项目将研究不可逆颜色转换的机制，从水母和珊瑚礁珊瑚中观察到一组荧光蛋白。这组蛋白质，被称为绿色到红色的光转换荧光蛋白（pcFPs），极大地推进了分子和细胞成像技术在生命科学。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，以成像超过可见光衍射极限的亚细胞事件。该项目的目标是揭示蛋白质运动和化学键重排之间的联系，这种相互作用最终导致了红色的形成。超快光谱方法和计算机建模将与pcfp的时变生化测量相结合。动力学和化学过程的关联将有助于阐明光生物学和酶学的新原理。这项工作将有利于培养本科生和研究生，以及培养博士后研究人员。此外，这个项目将成为若干外展活动的基石。为了加强基于证据的教学实践，ppcfp的前沿研究将整合到正式的课程中。这项工作的总体目标是研究蛋白质活性位点的超快原子运动如何与远程动力学和催化功能相关联。为了建立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FSRS是唯一可以跟踪从光激发开始的键振动的桌面技术。机械假设将通过评估速率和平衡常数的pcfp工程来测试，以承受修改的构建块。通过这种协同方法，将开发功能模型，集成超快原子运动，局部侧链旋转和整体链动力学在颜色修饰途径上。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.