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Probing multiple enzymatic methylation events in real time with NMR spectroscopy
Biophysical Journal ( IF 3.2 ) Pub Date : 2021-09-28 , DOI: 10.1016/j.bpj.2021.09.034
Emery T Usher 1 , Kevin E W Namitz 2 , Michael S Cosgrove 3 , Scott A Showalter 4
Affiliation  

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.



中文翻译:


使用核磁共振波谱实时探测多个酶促甲基化事件



蛋白质的翻译后修饰 (PTM) 对于真核生物中许多细胞过程的调节至关重要。研究最深入的蛋白质 PTM 之一是甲基化,其中酶催化甲基从辅因子转移到赖氨酸或精氨酸侧链。赖氨酸甲基化在组蛋白尾部尤其丰富,是表示活性或抑制基因的重要标记。鉴于甲基转移酶与转录调控的相关性,通过体外实验研究甲基转移酶功能是了解受调控基因表达的复杂机制的重要基石。迄今为止,大多数甲基转移酶表征策略依赖于放射性辅因子的使用、甲基转移副产物的检测或不连续型测定。尽管此类方法适用于某些应用,但有关多个甲基化事件和动力学中间体的信息经常丢失。在此,我们描述了使用二维 NMR 监测单个反应管中的单甲基化、二甲基化和三甲基化。为此,我们将13 C 掺入酶辅因子 S-腺苷甲硫氨酸的供体甲基中。通过这种方式,我们可以通过监测与单、二或三甲基赖氨酸相对应的不同共振的出现来研究酶促甲基化,而不需要同位素富集底物。为了证明该方法的功能,我们评估了三种赖氨酸甲基转移酶 Set7、MWRAD 2 (MLL1 复合物)和 PRDM9 对组蛋白 H3 尾部的活性。 我们通过连续的短二维异核单量子相干实验监测组蛋白 H3 尾部赖氨酸 4 的单甲基化或多甲基化,并将所得进展曲线拟合到一级动力学模型。总之,使用简单的辅助因子同位素富集在一锅实时反应中对 PTM 进行 NMR 检测,有望作为一种了解甲基转移酶和其他酶的复杂机制的方法。

更新日期:2021-11-03
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