Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States.
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, United States.
Methods. 2023 Oct;218:72-83. doi: 10.1016/j.ymeth.2023.07.010. Epub 2023 Jul 29.
Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [H, C]-HSQC and C direct-detect NMR measurements. Finally, we present C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.
翻译后修饰（PTMs）是可逆的化学修饰，可调节蛋白质的结构和功能。甲基化和乙酰化是两种此类PTM，具有不可或缺且特征明确的生物学作用，包括对染色质结构的调节；以及一些未知或了解甚少的作用，例如这些PTM对转录因子结构和功能的影响。对这些PTM功能的生物学见解的需求推动了一种无损且无标记方法的开发，该方法能够探究分子机制。在此，我们展示了一种实施核磁共振（NMR）方法的方案，该方法能够明确检测甲基化和乙酰化事件，并通过观察组蛋白H3尾部上的这些标记作为模型系统来证明其效用。我们利用辅因子和肽的策略性同位素富集，通过[H, C]-HSQC和C直接检测NMR测量进行可视化。最后，我们展示了有助于一维NMR实验的C标记方案，该方案相对于二维光谱减少了测量时间，同时对否则会造成光谱拥挤或限制低丰度分析物检测的背景信号进行了强大的过滤。
