Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to −1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein–DNA and protein–protein interactions in biological processes from replication to ubiquitin signalling.

翻译后修饰 (PTM) 动态调节细胞过程。赖氨酸经历一系列酰化，包括丙二酰化、琥珀酰化 (SucK) 和戊二酰化 (GluK)。这些 PTM 会增加赖氨酸侧链的大小，并在生理条件下将其电荷从 +1 反转为 -1，可能会影响蛋白质的结构和功能。为了了解这些 PTM 的功能作用，生化研究需要均质修饰的蛋白质。虽然通过遗传密码扩展对 PTM 及其模拟物进行位点特异性编码有助于表征许多 PTM 的功能作用，但带负电荷的赖氨酸酰化却无法实现这种方法。在这里，我们描述了通过硫酯衍生物暂时掩盖其负电荷，将 SucK 和 GluK 定点掺入蛋白质中。我们制备琥珀酰化和戊二酰化细菌和哺乳动物靶蛋白，包括不可重折叠的多结构域蛋白。这使我们能够研究琥珀酰化和戊二酰化如何影响代谢酶的酶活性，并调节从复制到泛素信号传导的生物过程中蛋白质-DNA 和蛋白质-蛋白质相互作用。