Fraser Olivia A, Dewing Sophia M, Usher Emery T, George Christy, Showalter Scott A
Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, United States.
Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Front Mol Biosci. 2023 Jan 6;9:1074743. doi: 10.3389/fmolb.2022.1074743. eCollection 2022.
Intrinsically disordered proteins are frequent targets for functional regulation through post-translational modification due to their high accessibility to modifying enzymes and the strong influence of changes in primary structure on their chemical properties. While lysine N-acetylation was first observed as a common modification of histone tails, proteomic data suggest that lysine acetylation is ubiquitous among both nuclear and cytosolic proteins. However, compared with our biophysical understanding of the other common post-translational modifications, mechanistic studies to document how lysine N-acetyl marks are placed, utilized to transduce signals, and eliminated when signals need to be turned off, have not kept pace with proteomic discoveries. Herein we report a nuclear magnetic resonance method to monitor N-lysine acetylation through enzymatic installation of aC-acetyl probe on a protein substrate, followed by detection through C direct-detect spectroscopy. We demonstrate the ease and utility of this method using histone H3 tail acetylation as a model. The clearest advantage to this method is that it requires no exogenous tags that would otherwise add steric bulk, change the chemical properties of the modified lysine, or generally interfere with downstream biochemical processes. The non-perturbing nature of this tagging method is beneficial for application in any system where changes to local structure and chemical properties beyond those imparted by lysine modification are unacceptable, including intrinsically disordered proteins, bromodomain containing protein complexes, and lysine deacetylase enzyme assays.
内在无序蛋白由于其对修饰酶具有高度可及性，且一级结构的变化对其化学性质有强烈影响，因而常常是翻译后修饰进行功能调控的靶点。虽然赖氨酸N - 乙酰化最初是作为组蛋白尾巴的常见修饰被观察到的，但蛋白质组学数据表明，赖氨酸乙酰化在核蛋白和胞质蛋白中都普遍存在。然而，与我们对其他常见翻译后修饰的生物物理理解相比，关于赖氨酸N - 乙酰标记如何被添加、如何用于转导信号以及在信号需要关闭时如何被去除的机制研究，尚未跟上蛋白质组学的发现。在此，我们报告一种核磁共振方法，通过在蛋白质底物上酶促安装碳 - 乙酰探针来监测N - 赖氨酸乙酰化，随后通过碳直接检测光谱进行检测。我们以组蛋白H3尾巴乙酰化为模型，展示了该方法的简便性和实用性。该方法最明显的优势在于它不需要额外的外源性标签，否则这些标签会增加空间位阻、改变修饰赖氨酸的化学性质，或者通常会干扰下游生化过程。这种标记方法的非干扰性质有利于应用于任何对赖氨酸修饰之外的局部结构和化学性质变化不可接受的系统，包括内在无序蛋白、含溴结构域的蛋白复合物以及赖氨酸脱乙酰酶酶活性测定。
