Imaging cotranslational protein folding with high spatiotemporal resolution in living cells

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Project Summary: Protein misfolding is implicated in many diseases, including cystic fibrosis, Alzheimer’s, Parkinson’s, and Huntington’s disease. Understanding protein folding mechanisms is therefore important for human health. While protein folding has been extensively studied for many years, the real-time folding of a protein has yet to be captured and quantified in a living cell due to a lack of adequate experimental spatiotemporal resolution. To fill the gap, I will combine single-molecule fluorescence microscopy and novel intrabodies that can distinguish unfolded and folded proteins to image cotranslational protein folding dynamics in living cells. With this technology, I propose to investigate the folding dynamics of the cystic fibrosis transmembrane conductance regulator (CFTR), the misfolding of which causes cystic fibrosis. CFTR is a good first application because it has already been demonstrated to predominantly fold cotranslationally in vitro. To characterize CFTR folding in living cells, I will develop genetically encodable intrabodies that bind folded and unfolded CFTR cytosolic domains (Aim 1). In parallel, I will establish methods to capture and quantify cotranslational protein folding dynamics using a model protein folding system based on GFP and its pre-existing intrabodies (Aim 2). With the technology from Aims 1 and 2 in hand (K99 phase), I will image CFTR cotranslational folding dynamics at the single mRNA level in living cells (Aim 3; R00 phase). This will reveal precisely when, where, and to what degree cotranslational CFTR folding is regulated within a fully natural context. Collectively, this work will not only shed new light on cotranslational protein folding dynamics, but will also lead to new strategies to combat cystic fibrosis and other protein misfolding diseases.