Collaborative Research: Dissecting photoconversion in fluorescent proteins frame by frame

本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颜色转换的机制。这组蛋白质，被称为绿色到红色的光转换荧光蛋白（pcFPs），在生命科学中极大地推进了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至关重要的作用，以使亚细胞事件超出可见光的衍射极限。该项目的目标是解开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红色的发展。超快光谱方法和计算机建模将与pcFP上的时间依赖性生化测量相结合。动力学和化学过程的相互关系将有助于阐明光生物学和酶学的新原理。 这项工作将促进本科生和研究生的培训以及博士后研究人员的培训。 此外，这一项目将成为若干外联活动的基石。 为了加强以证据为基础的教学实践，对pcFP的前沿研究将被纳入正式的课程。这项工作的首要目标是研究蛋白质活性位点中的超快原子运动如何与长程动力学和催化功能相关联。 为了开发一种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）将与光转换动力学测量和分子动力学模拟相结合。 目前，波长可调FSRS是唯一的桌面技术，可以跟踪键振动开始与光激发。 通过评估工程化以承载修饰的结构单元的pcFP的速率和平衡常数来测试机制假设。 通过这种协同方法，将开发功能模型，将超快原子运动，局部侧链旋转和全局链动力学整合到颜色修饰的途径中。该奖项反映了NSF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.