Event

The protein MYC is an intrinsically disordered transcription factor that is upregulated in >50% of cancers and engages in numerous protein-protein interactions [1]. These interactions are often regulated through posttranslational modifications (PTMs) within MYC’s N-terminus (transactivation domain), most commonly (poly)phosphorylation. Studying these interactions on a molecular level requires proteins with unique and defined PTM patterns, which are challenging to obtain by recombinant methods [2]. Flow-based solid-phase peptide synthesis (SPPS) could therefore be used to obtain long, uniquely modified MYC peptides to study PTM-dependent binding interactions on a molecular level [3,4].

A challenge that arose during the chemical synthesis of MYC’s transactivation domain—that is often encountered in SPPS—was the aggregation of growing peptide chains ("difficult sequences”), which can lead to incomplete couplings. Previous research into this sequence-dependent phenomenon was limited by the lack of high-throughput analytical methods, thus impeding systematic analysis. While flow-based SPPS allows for aggregation detection, it has so far not led to the development of tools for its suppression.

To enable the synthesis of MYC’s transactivation domain, we developed a “Synthesis Tag” (SynTag) consisting of six arginines connected via a cleavable MeDbz linker [5]. SynTag effectively improves batch- and flow-SPPS of “difficult sequences”, enhances the solubility of the cleaved peptides, and provides direct access to native sequences by hydrolysis, or peptide thioesters for Native Chemical Ligation (NCL). We demonstrate its utility in the first chemical synthesis of the MYC transactivation domain with a single NCL. We envisage SynTag to become a broadly applicable tool that enables the synthesis and study of previously unattainable peptides and proteins.

References:

[1]     C. Lourenco, D. Resetca, C. Redel, P. Lin, A. S. MacDonald, R. Ciaccio, T. M. G. Kenney, Y. Wei, D. W. Andrews, M. Sunnerhagen, C. H. Arrowsmith, B. Raught, L. Z. Penn, Nat. Rev. Cancer 2021, 21, 579–591

[2]     A. C. Conibear, Nat. Rev. Chem. 2020, 4, 674–695; T. Bilbrough, E. Piemontese, O. Seitz, Chem. Soc. Rev. 2022, 51, 5691–5730

[3]     N. Hartrampf, A. Saebi, M. Poskus, Z. P. Gates, A. J. Callahan, A. E. Cowfer, S. Hanna, S. Antilla, C. K. Schissel, A. J. Quartararo, X. Ye, A. J. Mijalis, M. D. Simon, A. Loas, S. Liu, C. Jessen, T. E. Nielsen, B. L. Pentelute, Science 2020, 368, 980.

[3]     E. T. Williams, K. Schiefelbein, M. Schuster, I. M. M. Ahmed, M. De Vries, R. Beveridge, O. Zerbe, N. Hartrampf, ChemRxiv 2024, 15, 8756–8765.

[4]     H. Bürgisser, E. T. Williams, R. Lescure, A. Premanand, A. Jeandin, N. Hartrampf, ChemRxiv 2023, DOI: 10.26434/chemrxiv-2023-7mz2c; in review.

Inquiries: nguyeny@sas.upenn.edu

Location: Carolyn Hoff Lynch Lecture Hall