Incorporating thioamides into proteins by native chemical ligation.

Autor:	Fiore KE; Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States., Phan HAT; Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States., Robkis DM; Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States; Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA, United States., Walters CR; Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States., Petersson EJ; Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: ejpetersson@sas.upenn.edu.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2021; Vol. 656, pp. 295-339. Date of Electronic Publication: 2021 May 07.
DOI:	10.1016/bs.mie.2021.04.011
Abstrakt:	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. (Copyright © 2021 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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