Collaborative Research: Dissecting photoconversion in fluorescent proteins frame by frame

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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.