Deciphering the Role of the Ser-Phosphorylation Pattern on the DNA-Binding Activity of Max Transcription Factor Using Chemical Protein Synthesis

Raj V. Nithun, Yumi Minyi Yao, Xiaoxi Lin, Shaimaa Habiballah, Ariel Afek*, Muhammad Jbara*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The chemical synthesis of site-specifically modified transcription factors (TFs) is a powerful method to investigate how post-translational modifications (PTMs) influence TF-DNA interactions and impact gene expression. Among these TFs, Max plays a pivotal role in controlling the expression of 15 % of the genome. The activity of Max is regulated by PTMs; Ser-phosphorylation at the N-terminus is considered one of the key regulatory mechanisms. In this study, we developed a practical synthetic strategy to prepare homogeneous full-length Max for the first time, to explore the impact of Max phosphorylation. We prepared a focused library of eight Max variants, with distinct modification patterns, including mono-phosphorylated, and doubly phosphorylated analogues at Ser2/Ser11 as well as fluorescently labeled variants through native chemical ligation. Through comprehensive DNA binding analyses, we discovered that the phosphorylation position plays a crucial role in the DNA-binding activity of Max. Furthermore, in vitro high-throughput analysis using DNA microarrays revealed that the N-terminus phosphorylation pattern does not interfere with the DNA sequence specificity of Max. Our work provides insights into the regulatory role of Max′s phosphorylation on the DNA interactions and sequence specificity, shedding light on how PTMs influence TF function.

Original languageEnglish
Article numbere202310913
JournalAngewandte Chemie - International Edition
Volume62
Issue number47
DOIs
StatePublished - 20 Nov 2023

Keywords

  • Chemical Protein Synthesis
  • Phosphorylation
  • Post-Translational Modifications
  • Total Synthesis
  • Transcription Factors

Fingerprint

Dive into the research topics of 'Deciphering the Role of the Ser-Phosphorylation Pattern on the DNA-Binding Activity of Max Transcription Factor Using Chemical Protein Synthesis'. Together they form a unique fingerprint.

Cite this