Applying Peptide and Protein Synthesis to Study Post-translational Modifications in Epigenetics and Beyond

Abstract

Epigenetics research focuses on the study of heritable gene regulatory mechanisms that do not involve changes of the DNA sequence. Such mechanisms include post-translational modifications of histone proteins that organize the genome in the nucleus into a nucleoprotein complex called chromatin, and which are of key importance in development and disease. Chemical biology tools as developed by my group, in particular synthetic peptide and protein chemistry, have been critical to elucidate epigenetic signaling mechanisms. As outlined below, they allow the reconstitution of chromatin carrying defined modifications and thus the elucidation of detailed molecular mechanisms.

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

“…2,10 The complex nature of PTM-mediated protein regulation is therefore difficult to investigate, and their study requires the production of proteins with specic PTM patterns. [11][12][13] Recombinant expression in conjunction with enzymatic modication (e.g. phosphorylation by kinases) can be used to obtain such proteins, but the precise control of location and number of PTMs is challenging.…”

“…However, the overall process of batch-SPPS and ligation is very time-consuming, laborious, and relatively lowyielding, impeding the production of large numbers of PTMpeptides and proteins. [11][12][13][14][15][16][17][18] In particular, researchers have long called for a general method for the synthesis of polyphosphorylated peptides with high yield and purity. 17 Recently, automated fast-ow peptide synthesis (AFPS) has proved successful for the rapid, linear synthesis of proteins exceeding 200 AA at a rate of approx.…”

Rapid Flow-Based Synthesis of Post-Translationally Modified Peptides and Proteins: A Case Study on MYC’s Transactivation Domain

“…2,10 The complex nature of PTM-mediated protein regulation is therefore difficult to investigate, and their study requires the production of proteins with specific PTM patterns. [11][12][13] Recombinant expression in conjunction with enzymatic modification (e.g. phosphorylation by kinases) can be used to obtain such proteins, but the precise control of location and Figure 1.…”

“…can be installed on a synthetic peptide sequence through either a building block method, or by late-stage modification of the full-length peptide or protein. [11][12][13][14][15][16][17][18] The former, and more popular, method has been exemplified in the successful production of PTM-peptides such as glycophosphonapeptide MYC[56-64], 19 cyclic diphosphorylated DSGFISK peptide, 20 and heptaphosphorylated Rho330-348 21 (Figure 1A), in which the PTM-amino acids were incorporated as building blocks during solution-or solid-phase peptide synthesis in batch (batch-SPPS). As batch-SPPS is typically limited to peptides of <50 amino acids (AA), native chemical ligation (NCL) or expressed protein ligation (EPL) are required to obtain longer sequences, 16,[22][23][24] as demonstrated for triphosphorylated HMGA1a 25 and phosphotyrosine-containing H2AY57p 26 (Figure 1A).…”

“…However, the overall process of batch-SPPS and ligation is very time-consuming, laborious, and relatively low yielding, limiting the efficient production of large numbers of PTM-peptides and proteins. [11][12][13][14][15][16][17][18] In particular, researchers have long called for a general method for the synthesis of polyphosphorylated peptides with high yield and purity. 17 Recently, automated fast-flow peptide synthesis (AFPS) has proved successful for the rapid, linear synthesis of proteins exceeding 200 AA at a rate of approx.…”