Reinert, Zachary
(2016)
Design of Unnatural Oligomers with Protein-like Tertiary Structure.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Proteins play key roles in biological processes that are highly dependent on their three-dimensional fold. Given the therapeutic relevance of many proteins, significant research effort has pursued the development of unnatural oligomers with protein-like folds. However, as the complexity of the target fold pushes beyond secondary structure, the difficulty of recapitulating the native fold becomes considerably high. There remains an unmet need to develop methods for tertiary structure mimicry of proteins on unnatural oligomers. Accessing complex protein-like folds on protease resistant backbones would yield improved therapeutics with high target specificity and sustained biological effects in vivo.
The goal of the current work was to generate design strategies for tertiary protein structure mimicry. We selected GB1, a protein that adopts a compact tertiary fold, as a system for backbone modification. We systematically replaced residues in the secondary structures of GB1 and measured the resulting changes to folding thermodynamics and structure. Combination of separate modifications into one protein led to a mutant that showed evidence for tertiary folding despite having an ~ 20% unnatural backbone sequence. Furthermore, grafting the combined backbone alterations onto a side-chain sequence that encodes for a more stable and identical tertiary fold resulted in a significant stabilization of the folded state. The observations supported a general design hypothesis that proteins have two mutually orthogonal design sequences: 1) backbone and 2) side-chain.
The detailed effects of unnatural residues on protein folding thermodynamics were also examined and revealed several interesting trends. A series of α→β3 substitutions were implemented in GB1. β3-Residues are less conformationally restricted than α-residues, yet they entropically stabilized protein folding. Rigidification of the backbone through either cyclization or Cα-methylation respectively somewhat or significantly stabilized the folded state.
We studied other types of unnatural residues in the context of a small hairpin peptide. γcyc-Residues were found to stabilize the hairpin fold greater than the natural backbone. However when applied to GB1, the same strategy was detrimental to folding. Optimization of the location of unnatural residues resulted in a restoration of near wild-type folded stability.
Overall the developed strategies should be applicable to larger, more complex protein folds.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Reinert, Zachary | zer4@pitt.edu | ZER4 | |
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| ETD Committee: |
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| Date: |
22 January 2016 |
| Date Type: |
Publication |
| Defense Date: |
12 November 2015 |
| Approval Date: |
22 January 2016 |
| Submission Date: |
23 September 2015 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
186 |
| 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: |
protein, foldamers, biophysics, unnatural residues, GB1 |
| Date Deposited: |
22 Jan 2016 15:27 |
| Last Modified: |
22 Jan 2017 06:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/26138 |
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