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Thioamide substitutions in the backbones of proteins can modulate their structure and thermostability, or serve as spectroscopic probes in fluorescence quenching experiments. Using native chemical ligation, we have produced the first examples of a protein (calmodulin) containing two thioamides. Dithioamide variants were made to explore the effects of combining stabilizing, neutral, and destabilizing single thioamide substitutions. One of the dithioamide calmodulin variants exhibited stabilization greater than any monothioamide variant, although the effect could not easily be anticipated from the results of single substitutions. Each of the calmodulin variants retained the ability to bind a target peptide, and the dithioamide proteins exhibited an increase in fluorescence quenching of tryptophan relative to their single thioamide counterparts. These results show that multiply thioamidated proteins can be synthesized, and that properly placed thioamides can be used to increase protein thermostability or enhance fluorecsence quenching in peptide binding experiments.
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Chapter Ten - Incorporating thioamides into proteins by native chemical ligation
The thioamide is a versatile replacement of the peptide backbone with altered hydrogen bonding and conformational preferences, as well the ability participate in energy and electron transfer processes. Semi-synthetic incorporation of a thioamide into a protein can be used to study protein folding or protein/protein interactions using these properties. Semi-synthesis also provides the opportunity to study the role of thioamides in natural proteins. Here we outline the semi-synthesis of a model protein, the B1 domain of protein G (GB1) with a thioamide at the N-terminus or the C-terminus. The thioamide is synthetically incorporated into a fragment by solid-phase peptide synthesis, whereas the remainder of the protein is recombinantly expressed. Then, the two fragments are joined by native chemical ligation. The explicit protocol for GB1 synthesis is accompanied by examples of applications with GB1 and other proteins in structural biology and protein misfolding studies.
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- Award ID(s):
- 1708759
- PAR ID:
- 10280363
- Editor(s):
- Petersson, E. J.
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 656
- ISSN:
- 1557-7988
- Page Range / eLocation ID:
- 295-339
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1016/bs.mie.2021.04.011
