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

Oregon State University, Corvallis OR

Investigators

Abstract

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.