Analyses of All Possible Point Mutations within a Protein Reveals Relationships between Function and Experimental Fitness: A Dissertation
Authors
Roscoe, Benjamin P.Faculty Advisor
Daniel N. Bolon, PhDAcademic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
Biochemistry and Molecular PharmacologyDocument Type
Doctoral DissertationPublication Date
2014-03-25Keywords
Dissertations, UMMSAmino Acid Sequence
Mutagenesis
Point Mutation
Sequence Analysis, Protein
Ubiquitin
Amino Acid Sequence
Mutagenesis
Point Mutation
Protein Sequence Analysis
Ubiquitin
Biochemistry
Cellular and Molecular Physiology
Molecular Biology
Molecular Genetics
Metadata
Show full item recordAbstract
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.DOI
10.13028/M2G027Permanent Link to this Item
http://hdl.handle.net/20.500.14038/32075Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/M2G027