This article describes a method for site-specific incorporation of succinylation (SucK) and glutarylation (GluK) into proteins using genetic code expansion. The researchers developed thioester derivatives of SucK and GluK that temporarily mask their negative charge, enabling their incorporation into proteins. These derivatives can be converted to their active forms through thioester hydrolysis, allowing the study of their functional roles. The approach was applied to bacterial and mammalian target proteins, including non-refoldable multidomain proteins, enabling the investigation of how these modifications affect enzymatic activity, protein–protein and protein–DNA interactions in biological processes such as replication and ubiquitin signaling. The study highlights the importance of acidic lysine acylations, including SucK and GluK, in regulating cellular functions. These modifications alter the charge and structure of lysine residues, potentially impacting protein function. The researchers demonstrated that these modifications can be recognized by endogenous deacylases, such as SIRT5 and CobB, and that their effects on protein function can be studied using biochemical and structural methods. The method allows for the generation of homogeneously modified proteins, which is essential for understanding the functional roles of these post-translational modifications. The study also shows that these modifications can influence metabolic enzymes, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and protein–protein interactions, such as those involving ubiquitin (Ub) and deubiquitylases (DUBs). The findings suggest that acidic lysine acylations play a significant role in regulating various biological processes, including DNA replication, DNA repair, and cell cycle control. The approach provides a general and robust tool for studying the impact of these modifications on protein structure and function.This article describes a method for site-specific incorporation of succinylation (SucK) and glutarylation (GluK) into proteins using genetic code expansion. The researchers developed thioester derivatives of SucK and GluK that temporarily mask their negative charge, enabling their incorporation into proteins. These derivatives can be converted to their active forms through thioester hydrolysis, allowing the study of their functional roles. The approach was applied to bacterial and mammalian target proteins, including non-refoldable multidomain proteins, enabling the investigation of how these modifications affect enzymatic activity, protein–protein and protein–DNA interactions in biological processes such as replication and ubiquitin signaling. The study highlights the importance of acidic lysine acylations, including SucK and GluK, in regulating cellular functions. These modifications alter the charge and structure of lysine residues, potentially impacting protein function. The researchers demonstrated that these modifications can be recognized by endogenous deacylases, such as SIRT5 and CobB, and that their effects on protein function can be studied using biochemical and structural methods. The method allows for the generation of homogeneously modified proteins, which is essential for understanding the functional roles of these post-translational modifications. The study also shows that these modifications can influence metabolic enzymes, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and protein–protein interactions, such as those involving ubiquitin (Ub) and deubiquitylases (DUBs). The findings suggest that acidic lysine acylations play a significant role in regulating various biological processes, including DNA replication, DNA repair, and cell cycle control. The approach provides a general and robust tool for studying the impact of these modifications on protein structure and function.