Issue 23, 2024

Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain

Abstract

Protein–protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (Nε-methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs.

Graphical abstract: Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain

Supplementary files

Article information

Article type
Edge Article
Submitted
21 1 2024
Accepted
04 5 2024
First published
07 5 2024
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2024,15, 8756-8765

Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain

E. T. Williams, K. Schiefelbein, M. Schuster, I. M. M. Ahmed, M. De Vries, R. Beveridge, O. Zerbe and N. Hartrampf, Chem. Sci., 2024, 15, 8756 DOI: 10.1039/D4SC00481G

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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