Lengyel, George
(2014)
Strategies for Peptide Backbone Modification in Protein Beta-Sheets.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Design of foldamers, unnatural backbone oligomers that mimic the structure of proteins, is an important field of research as these species can bind to natural proteins but are resistant to proteolytic degradation. We have focused on developing strategies for the design of unnatural oligomers that adopt β-sheet secondary structures like those commonly found in protein tertiary folds. Our approach is to modify natural peptide sequences that encode for β-sheet folds with various unnatural amino acid building blocks to produce hybrid-backbone peptides that fold like the parent sequence in aqueous solution.
Through evaluation of β-hairpin model systems using multidimensional NMR, we have discovered several design strategies that may be applicable to mimicry of sheets found in larger protein tertiary structures and have ranked unnatural monomer types in order of increasing sheet propensity: β-residue < N-methyl-residue ≤ vinylogous γ4-residue < cyclic γ-residue. These substitutions require a 2:2 or 2:1 α- to β-residue substitution or 1:1 α- to γ- or α- to N-methyl-residue substitution in order to maintain native-like folding behavior.
We applied these unnatural residue substitutions in protein GB1, a 56 residue protein with a complex tertiary fold consisting of a four stranded β-sheet packed against an α-helix. Using thermal denaturation melts and circular dichroism spectroscopy, we have determined that the trend of sheet propensity seen in the hairpin peptide is similar in a tertiary fold with the caveat that the position of the unnatural residues matters greatly. Substitution strategies that lengthen the strands of the β-sheet have varying effects on the stability of the folded structure depending on their placement; substitutions near the center of the strands are significantly more destabilizing than those placed near the termini. Use of N¬-methylated residues is not limited in this fashion, but their positioning must be chosen so as to avoid disruption of inter-strand hydrogen bonding.
Overall, we have determined that several unnatural residue types can be used to promote sheet formation with limited destabilization; these residues could potentially be used in other proteins with tertiary folded structures.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
24 September 2014 |
| Date Type: |
Publication |
| Defense Date: |
16 April 2014 |
| Approval Date: |
24 September 2014 |
| Submission Date: |
31 March 2014 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
297 |
| 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: |
peptide, foldamer, protein, backbone, unnatural residue |
| Date Deposited: |
24 Sep 2014 13:53 |
| Last Modified: |
15 Nov 2016 14:18 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/20904 |
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