CAREER: Directly Visualizing the Conformational Dynamics and Non-covalent Interactions of Biological Macromolecules in Living Cells using Fast Raman Relaxation Imaging

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor David Punihaole of the University of Vermont is studying the development of new chemical imaging methods to investigate protein folding in living cells. Proteins must fold into specific 3D shapes to function correctly, but most current tools can only observe this process in test tubes, not within cells. To overcome this challenge, this project seeks to create a technique called Fast Raman Relaxation Imaging (FRReI), which will track protein folding in real time inside cells. This technique seeks to transform the current understanding of how cells regulate protein folding, preventing misfolding linked to diseases and genetic disorders. The project also includes an educational program that partners with the Library of Congress to train undergraduate students in advanced imaging techniques. This program seeks to equip students with valuable skills for future careers in science and technology. This project seeks to develop Fast Raman Relaxation Imaging (FRReI), a novel technique to directly monitor protein folding structural dynamics in living cells. The specific research objectives of this project are: (1) Build and validate the FRReI platform by incorporating an IR laser line into a stimulated Raman microscope; (2) Create novel Raman probes to attach to proteins for sensitive monitoring of folding dynamics; and (3) Use FRReI to study how different cellular environments, like the cytosol and mitochondria, influence protein folding. Success in this project could provide transformative insights into how cells regulate protein folding through mechanisms like post-translational modifications, molecular crowding, spatial compartmentalization, and chaperones. 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.