GSBS Dissertations and Theses

Date of Completion

3-25-2014

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Biochemistry and Molecular Pharmacology

Subjects

Dissertations, UMMS; Amino Acid Sequence; Mutagenesis; Point Mutation; Sequence Analysis, Protein; Ubiquitin

Abstract

The primary amino acid sequence of a protein governs its specific cellular functions. Since the cracking of the genetic code in the late 1950’s, it has been possible to predict the amino acid sequence of a given protein from the DNA sequence of a gene. Nevertheless, the ability to predict a protein’s function from its primary sequence remains a great challenge in biology. In order to address this problem, we combined recent advances in next generation sequencing technologies with systematic mutagenesis strategies to assess the function of thousands of protein variants in a single experiment. Using this strategy, my dissertation describes the effects of most possible single point mutants in the multifunctional Ubiquitin protein in yeast. The effects of these mutants on the essential activation of ubiquitin by the ubiquitin activating protein (E1, Uba1p) as well as their effects on overall yeast growth were measured. Ubiquitin mutants defective for E1 activation were found to correlate with growth defects, although in a non-linear fashion. Further examination of select point mutants indicated that E1 activation deficiencies predict downstream defects in Ubiquitin function, resulting in the observed growth phenotypes. These results indicate that there may be selective pressure for the activity of the E1enzyme to selectively activate ubiquitin protein variants that do not result in functional downstream defects. Additionally, I will describe the use of similar techniques to discover drug resistant mutants of the oncogenic protein BRAFV600E in human melanoma cell lines as an example of the widespread applicability of our strategy for addressing the relationship between protein function and biological fitness.

Rights and Permissions

Copyright is held by the author, with all rights reserved.

TableS2_1.xlsx (255 kB)
Number of illumina sequence reads for each mutant during bulk Fitness competition

TableS2_2.xlsx (63 kB)
Relative effects of amino acid substitutions on yeast growth rate

TableS2_3.xlsx (156 kB)
Sequencing counts of mutant reads in the plasmid library and after outgrowth in galactose media with WT ubiquitin co-expression

TableS2_4.xlsx (51 kB)
Change in solvent accessible surface area (d-ASA) of 123 ubiquitin chains in 44 heterocomplexes and the 2O6V.PDB homotetramer

TableS2_5.xlsx (11 kB)
Fitness measurements and changes in free energy of folded states Relative to unfolded states compiled in the literature

TableS3_1.xlsx (208 kB)
Sequencing counts

TableS3_2.xlsx (100 kB)
E1 reactivity estimates from bulk competitions

TableS3_3.xlsx (59 kB)
Sequencing counts of E+H region mutants reacted with excess E1

TableS3_4.xlsx (35 kB)
E1 reactivity estimates from bulk competitions with excess E1

TableS5_1.xlsx (271 kB)
Number of illumina sequence reads for each ubiquitin point mutants during competitive growth at 36º C

TableS5_2.xlsx (62 kB)
Relative growth rate of ubiquitin point mutants at 36º C

 
 

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