Luo, Ji
(2020)
OPTICAL AND SMALL-MOLECULE CONTROL OF PROTEIN FUNCTION THROUGH GENETIC CODE EXPANSION.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Expanding the genetic code for site-specific incorporation of various biophysical probes and labels, such as fluorescent probes, photolabile caging groups, optical probes, photoswitches, bioorthogonal chemselective groups, offers a sophisticated tool for exploring protein structure and function, dissecting the cellular processes, and developing proteins with novel properties. Site-specific installation of distinct functional groups on a protein of interest is achieved using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in response to an amber stop codon (UAG) placed in the gene of interest. In this dissertation, genetic encoding of a series of diverse unnatural amino acids (UAAs), including photocaged lysines, coumarin lysines, phthalimide lysines, photocaged cysteines, photocaged tyrosines, photoisomerizable phenylalanines, and azido lysines, were achieved through the discovery of the engineered pyrrolysyl-tRNA synthetase/tRNA pair from bacteria to eukaryotic cells. Strategic placement of these functional groups renders the protein inactive until deprotection through a bioorthogonal external trigger delivers the active wild-type protein. This developed methodology enables the conditional control of various cellular processes with spatiotemporal precision in a non-invasive way, including enzymatic activation, protein folding, protein translocation, gene repair (UvrD), post-translational modification (SUMO1), DNA recombination (Cre), RNA processing (Csy4), and gene editing (CRISPR/Cas9).
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
| Title | Member | Email Address | Pitt Username | ORCID |
|---|
| Committee Chair | Deiters, Alexander | | | | | Committee Member | Childers, W. Seth | | | | | Committee Member | Islam, Kabirul | | | | | Committee Member | Van Houten, Bennett | | | |
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| Date: |
16 January 2020 |
| Date Type: |
Publication |
| Defense Date: |
25 September 2017 |
| Approval Date: |
16 January 2020 |
| Submission Date: |
6 January 2020 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
335 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
genetic code expansion, unnatural amino acid, protein function |
| Date Deposited: |
16 Jan 2020 18:59 |
| Last Modified: |
16 Jan 2022 06:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/38097 |
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