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

Williams, Elyse T. and Schiefelbein, Kevin and Schuster, Matthias and Ahmed, Ikhlas M. M. and De vries, Marije and Beveridge, Rebecca and Zerbe, Oliver and Hartrampf, Nina (2024) Rapid flow-based synthesis of post-translationally modified peptides and proteins : a case study on MYC's transactivation domain. Chemical Science. ISSN 2041-6539 (https://doi.org/10.1039/D4SC00481G)

[thumbnail of Williams-etal-CS-2024-Rapid-flow-based-synthesis-of-post-translationally-modified-peptides-and-proteins]
Preview
Text. Filename: Williams-etal-CS-2024-Rapid-flow-based-synthesis-of-post-translationally-modified-peptides-and-proteins.pdf
Final Published Version
License: Creative Commons Attribution 3.0 logo

Download (2MB)| Preview

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.