Analyses of the effects of all ubiquitin point mutants on yeast growth rate
Program in Bioinformatics and Integrative Biology; Department of Biochemistry and Molecular Pharmacology
Amino Acid Substitution; DNA Mutational Analysis; Microbial Viability; Models, Molecular; Mutant Proteins; *Point Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Molecular Biology
The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin, almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent-inaccessible core of ubiquitin, all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that, for these mutants, subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.
DOI of Published Version
J Mol Biol. 2013 Apr 26;425(8):1363-77. doi: 10.1016/j.jmb.2013.01.032. Link to article on publisher's site
Journal of molecular biology
Roscoe, Benjamin P.; Thayer, Kelly M.; Zeldovich, Konstantin B.; Fushman, David; and Bolon, Daniel N. A., "Analyses of the effects of all ubiquitin point mutants on yeast growth rate" (2013). University of Massachusetts Medical School Faculty Publications. 74.