Research Advance: Extending chemical perturbations of the Ubiquitin fitness landscape in a classroom setting

Abstract

150 words) 21 Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary 22 time, it is highly tolerant to mutation during selection experiments performed in the laboratory. 23 We have proposed that this discrepancy results from the difference between fitness under 24 laboratory culture conditions and the selective pressures in changing environments over 25 evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep 26 mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-27 fluorocytosine, Amphotericin B, CaCl 2 , Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, 28 p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational 29sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In 30 total, our experiments have uncovered a sensitizing condition for every position in Ub except 31 Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical 32 constraints, our work helps to resolve the inconsistencies between deep mutational scanning 33 experiments and sequence conservation over evolutionary timescales.