Heath, Shelby
(2023)
SYNTHETIC APPROACHES TOWARDS THE DEVELOPMENT OF PROTEIN
MIMETICS AND THE STUDY OF POST-TRANSLATIONAL CYCLIC IMIDE
FORMATION.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Proteins adopt complex folded structures that have evolved to perform specific functions. Naturally occurring proteins are synthesized using a toolbox of 20 L--amino acids that are linked together in various combinations, resulting in a finite number of chemically distinct proteins. Post-translational modifications (PTMs) are covalent alterations that nature uses to exponentially increase the number of chemically distinct proteins that define the complex web of the proteome. While nature has developed PTMs that are required for biological function, the accumulation of age-related PTMs can inhibit the cell’s ability to maintain homeostasis.
This work primarily focuses on the use of protein chemical synthesis as a tool to study the effects of Asn deamidation and Asp isomerization, an age-related PTM, on protein structure and stability. Using chemical synthesis, we have demonstrated the ability to incorporate isomerized Asp residues into a full-length protein. Through the successful synthesis of proteins containing isomeric Asp residues, we were able to determine the impacts of Asp isomerization on protein folded structure and folding thermodynamics of the B1 domain of Streptococcal protein G.
To further enhance understanding of Asp isomerization, we sought to develop a chemical tool to generate the cyclic imide intermediate of the isomerization pathway. Using a photo-cleavable amide protecting group coupled with a labile ester leaving group, we have demonstrated the ability to generate the imide in situ under mild conditions. This work presents a potentially useful method for the study of Asp isomerization following imide hydrolysis in biologically relevant proteins.
Additionally, utilizing protein chemical synthesis, we explored the effects of backbone modifications on protein-protein interactions (PPIs). The field of proteomimetics has historically utilized noncanonical amino acids towards the development of artificial scaffolds that retain native-like folded structure and function. To add to this body of knowledge, we sought to apply proteomimetic methods towards the development of peptide tertiary structure mimics that retain the ability to engage a binding partner. While we were unsuccessful in this endeavor, we were able to show the negative impact of even subtle alteration of side chain display on a weak affinity PPI.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
6 September 2023 |
| Date Type: |
Publication |
| Defense Date: |
1 August 2023 |
| Approval Date: |
6 September 2023 |
| Submission Date: |
3 August 2023 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
164 |
| 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: |
peptidomimetics
heterogeneous backbone
inhibitor
deamidation
isomerization |
| Date Deposited: |
06 Sep 2023 16:13 |
| Last Modified: |
06 Sep 2023 16:13 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/45234 |
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