Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain
Elyse T. Williams, Kevin Schiefelbein, Matthias Schuster, Ikhlas M. M. Ahmed, Marije De Vries, Rebecca Beveridge, Oliver Zerbe, Nina Hartrampf
Abstract
-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.