Protein–protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (N^ε-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.

中文翻译：

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基于流程的快速翻译后修饰肽和蛋白质合成：MYC 反式激活结构域的案例研究

c-Myc (MYC) 的蛋白质-蛋白质相互作用通常受到翻译后修饰 (PTM) 的调节，例如磷酸化及其串扰。研究这些相互作用需要具有独特 PTM 模式的蛋白质，而通过重组方法获得这些蛋白质具有挑战性。标准肽合成和天然化学连接可以产生此类修饰蛋白，但非常耗时，因此通常仅限于单个 PTM 的研究。在此，我们报告了基于流的方法的开发，用于快速合成磷酸化 MYC 序列（最多 84 个 AA），并证明了该方法在掺入其他 PTM（N ^ε -甲基化、硫酸化、乙酰化、糖基化）方面的多功能性。 ）及其组合。成功制备并以高产率和纯度分离出含有多达 7 个 PTM 和多达 5 个位点磷酸化的肽。我们进一步生产了十种 PTM 修饰的 MYC 反式激活结构域 (TAD) 类似物，以使用异核 NMR 和天然质谱法筛选与肿瘤抑制蛋白 Bin1 的结合。我们确定了磷酸化和糖基化对 MYC:Bin1 相互作用强度的影响，并揭示了 MYC 序列长度对结合的影响。我们的平台可快速合成多达 84 个 AA 且具有独特 PTM 模式的 MYC 序列，从而能够在分子水平上系统研究 PTM 功能，并为快速筛选构建体提供便捷的方法。