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Author ORCID Identifier

https://orcid.org/0000-0001-7952-1551

AccessType

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2020

Month Degree Awarded

September

First Advisor

Eric Strieter

Subject Categories

Chemistry | Life Sciences

Abstract

Ubiquitin is a 76 amino acids protein that is evolutionary conserved in eukaryotes. It is an important signaling molecule in a plethora biological events, such as protein degradation, DNA damage response, and transcription. This thesis aims to develop engineered protein as a tool to study ubiquitin signaling. Through targeted mutagenesis and directed evolution, a deubiquitinase is reprogrammed into a transamidase, which lead to the generation of ubiquitinprotein conjugates with discrete ubiquitin linkages through auto-ubiquitination. These ubiquitin-protein conjugates could be used as a model substrate to profile their interaction of different ubiquitin interacting proteins. In addition, using directed evolution and deep sequencing, a nanobody-based binder targeting a deubiquitinase is engineered. This nanobody could be utilized as an intracellular probe to study the regulation of the deubiquitinase with great spatial and temporal control.

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