Protein Folding from Solution to the Living Cell

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Intellectual merit:For many years research on the dynamic behavior of proteins has focused on isolated proteins manipulated outside of the cellular environment (in vitro). There are only a few systems in which investigators have moved into more complex environments, including the living cell (in vivo). The proposed studies will develop Fast Relaxation Imaging, a technique that looks at biomolecular dynamics inside living cells, allowing connections to be made between what has been learned in vitro to how proteins behave in vivo. In the proposed experiments a small laser-induced temperature jump initiates protein folding kinetics, protein-protein interactions, or nucleic acid dynamics inside cells. A fluorescence microscope images the resulting dynamics as a function of time, making a "folding movie" inside the cell. The experiment will be applied to study the heat shock response of cells, interactions of the protein alpha-synuclein with cell membranes, and folding of metabolic and signal enzymes in various compartments of different cell types. Broader Impacts:Because protein dynamics are critical to protein function, understanding how cells modulate the stability and kinetics of their constituent biomolecules is critical to understanding cell function and the regulation of cellular processes such as gene transcription, apoptosis and cell division. Defining protein behavior in the cell will also lead to determining what aspects of modulation are evolved and have been part of the selection process on protein structure. The research is complemented by several outreach programs, including recruitment of summer undergraduate research students, an exchange and teaching program with Hanoi University of Science to bring their teaching and research up to modern standards, and continuation of a K-12 outreach program that was set up by a biophysics student in the PIs lab, and is now available as a web-based tutorial in biophysics: a student, with guidance of a faculty mentor, collaborates with middle- and high school teachers to help them create educational materials employing biophysical visualization tools. The broader impacts also include development of the instrumentation for the proposed experiments. This instrumentation could be become widely used in other laboratories.This project is jointly funded by Cellular Systems and Molecular Biophysics in MCB.