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Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance

2018; The Company of Biologists; Volume: 7; Issue: 7 Linguagem: Inglês

10.1242/bio.036103

2046-6390

David Mavor, Kyle A. Barlow, Daniel Asarnow, Yuliya Birman, Derek Britain, Weilin Chen, Evan M. Green, Lukas Kenner, Bruk Mensa, Leanna S. Morinishi, Charlotte Nelson, Erin M. Poss, Pooja Suresh, Ruilin Tian, Taylor Arhar, Beatrice Ary, David Bauer, Ian D. Bergman, Rachel M. Brunetti, Cynthia M. Chio, Shizhong Dai, Miles Sasha Dickinson, Susanna K. Elledge, Cole Helsell, Nathan L. Hendel, Emily L. Kang, Nadja Kern, Matvei Khoroshkin, Lisa L. Kirkemo, Greyson R. Lewis, Kevin Lou, Wesley M. Marin, Alison M. Maxwell, Peter F. McTigue, Douglas Myers-Turnbull, Tamas L Nagy, Andrew M. Natale, Keely Oltion, Sergei Pourmal, Gabriel K. Reder, Nicholas J. Rettko, Peter J. Rohweder, Daniel M. C. Schwarz, Sophia K. Tan, Paul V. Thomas, Ryan W. Tibble, Jason P. Town, Mary K. Tsai, Fatima S. Ugur, Douglas R. Wassarman, Alexander M. Wolff, Taiasean Wu, Derek Bogdanoff, Jennifer Li, Kurt S. Thorn, Shane Ó’Conchúir, Danielle L. Swaney, Eric D. Chow, Hiten D. Madhani, Sy Redding, Daniel N. Bolon, Tanja Kortemme, Joseph L. DeRisi, Martin Kampmann, James S. Fraser,

vaccines and immunoinformatics approaches

ABSTRACT Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.