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Strategies for Peptide Backbone Modification in Protein Beta-Sheets

Lengyel, George (2014) Strategies for Peptide Backbone Modification in Protein Beta-Sheets. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Lengyel, Georgegal23@pitt.eduGAL23
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairHorne, William S.horne@pitt.eduHORNE
Committee MemberCurran, Dennis P.curran@pitt.eduCURRAN
Committee MemberWeber, Stevesweber@pitt.eduSWEBER
Committee MemberWetzel, Ronaldrwetzel@pitt.eduRWETZEL
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


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