Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
J Biomol NMR. 2013 Jan;55(1):97-104. doi: 10.1007/s10858-012-9690-x. Epub 2012 Nov 29.
Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D(2)O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium. To encourage the conversion by serine hydroxymethyltransferase, we performed the cell-free reaction without the addition of any glycine, exploiting the capability of the enzyme to convert serine to glycine with the help of tetrahydrofolate. (13)C-HSQC spectra of ubiquitin produced with (13)C/(15)N-serine showed that about a quarter of the glycine residues derived from serine were stereospecifically deuterated. Pulse sequences are presented that select the signals from the stereospecifically deuterated glycine residues.
甘氨酸的α-质子的立体专一性共振分配通常难以通过测量标量耦合常数或核 Overhauser 效应来获得。在这里，我们表明这些立体专一性共振分配可以通过 D(2)O 中的无细胞蛋白质合成轻松获得，因为丝氨酸羟甲基转移酶天然存在于大肠杆菌细胞提取物中，它选择性地用氘取代甘氨酸的 pro-2S 质子。为了鼓励丝氨酸羟甲基转移酶进行转化，我们在没有添加任何甘氨酸的情况下进行无细胞反应，利用酶在四氢叶酸的帮助下将丝氨酸转化为甘氨酸的能力。用 (13)C/(15)N-丝氨酸合成的泛素的 (13)C-HSQC 谱表明，大约四分之一来源于丝氨酸的甘氨酸残基是立体专一性氘化的。本文介绍了选择来自立体专一性氘化甘氨酸残基的信号的脉冲序列。
