1 PROJECT SUMMARY 2 Cystic fibrosis (CF) is a lethal genetic lung disease caused by mutations in the Cystic Fibrosis 3 Transmembrane Conductance Regulator (CFTR), an epithelial anion channel protein. The most common patient 4 mutation, deletion of phenylalanine 508 (ΔF508) and many of over 1000 mutations destabilize CFTR. Unstable 5 ΔF508 is recognized by molecular chaperones as unfolded. Chaperone binding to mutant CFTR eventually 6 results in pre-mature degradation leading to CF. Precisely how molecular chaperones recognize ΔF508 CFTR 7 as unfolded is unclear. Previous in vitro studies characterized chaperone binding hotspots in CFTR peptides and 8 individual domains; however, the chaperone binding hotspots that ΔF508 unfolds and exposes in the cell remains 9 unknown. We hypothesize domain and sub-domain level ΔF508 unfolding exposes chaperone binding hotspots 10 recognized by molecular chaperones. Additionally, we hypothesize stabilizing ΔF508 CFTR with FDA approved 11 CF therapies will restore domain and sub-domain level chaperone recognition towards WT CFTR. In aim I, we 12 propose simulating full-length WT and ΔF508 CFTR structures in silico to determine how ΔF508 deviates from 13 normal WT structure. We can build ΔF508 CFTR models in Rosetta, dock CF drugs to the structures, and 14 benchmark methods for sampling CFTR conformational space by comparing simulations results to published 15 experimental data. In aim II, we propose site-specific non-canonical amino acid incorporation of photochemical 16 crosslinkers to covalently capture molecular chaperone binding to CFTR domains and sub-domains in live cells. 17 We can identify and quantify site-specific CFTR interactors by affinity-purification mass spectrometry with 18 Tandem Mass Tag labeling. Furthermore, we will again stabilize ΔF508 CFTR with CF drugs to examine how 19 small molecule binding impacts molecular chaperone binding. Studying the relationship between drug binding 20 and chaperone recognition is important because the only targeted treatment for CF involved stabilizing ΔF508 21 CFTR with small molecules called pharmacological chaperones. However, pharmacological chaperones are 22 discovered through expensive phenotypic screens and their molecular mechanisms remain unclear. We seek to 23 distinguish whether pharmacological chaperones change molecular chaperone recognition in the domain of 24 binding or nearby domains through allosteric effects. Other misfolding diseases, such as cardiac arrythmia Long 25 QT Syndrome, are caused by mutations in similar membrane proteins, but drug treatments lay out of reach due 26 to lack of assays for screening. Thus, we developed methods to evaluate pharmacological chaperones and their 27 contributions to CFTR structural stability. Our novel approach will elucidate the interplay between unstable 28 mutants, molecular chaperone recognition, and pharmacological chaperone rescue by leveraging and integrating 29 data from computational struct...