Presented By: Department of Chemistry
New tools for flow-based peptide and protein synthesis
Nina Hartrampf (University of Zurich)
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.
[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, DOI: 10.26434/chemrxiv-2024-mfpkx.
[4] H. Bürgisser, E. T. Williams, R. Lescure, A. Premanand, A. Jeandin, N. Hartrampf, ChemRxiv
2023, DOI: 10.26434/chemrxiv-2023-7mz2c.
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.
[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, DOI: 10.26434/chemrxiv-2024-mfpkx.
[4] H. Bürgisser, E. T. Williams, R. Lescure, A. Premanand, A. Jeandin, N. Hartrampf, ChemRxiv
2023, DOI: 10.26434/chemrxiv-2023-7mz2c.
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