Genetic Code Expansion Tools to Study Lysine Acylation

Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function, such as the activity, localization, and stability of enzymes. Mass spectrometric identification of lysine acylations has witnessed tremendous improvements in sensitivity over the last decade, facilitating the discovery of thousands of lysine acylation sites in proteins involved in all essential cellular functions across organisms of all domains of life. However, the vast majority of currently known acylation sites are of unknown function. Semi‐synthetic methods for installing lysine derivatives are ideally suited for in vitro experiments, while genetic code expansion (GCE) allows the installation and study of such lysine modifications, especially their dynamic properties, in vivo. An overview of the current state of the art is provided, and its potential is illustrated with case studies from recent literature. These include the application of engineered enzymes and GCE to install lysine modifications or photoactivatable crosslinker amino acids. Their use in the context of central metabolism, bacterial and viral pathogenicity, the cytoskeleton and chromatin dynamics, is investigated. Genetic code expansion facilitates the production of proteins containing defined lysine acylations. These proteins can then be used to study the impact of the modification on their properties in vitro or on the physiology of the cell. This review summarizes recent technological developments and provides illustrative examples of their application.