Sirtuin-Derived Covalent Binder for the Selective Recognition of Protein Crotonylation

Lysine crotonylation (Kcr) is increasingly recognized as a key protein post-translational modification. However, selective detection and enrichment of crotonylated proteins remains a challenging task. Herein we present a covalent binder for the selective recognition of protein crotonylation. Based on proximity-induced crosslinking, a bacterial sirtuin (CobB) was remodeled with genetically installed thiol-bearing noncanonical amino acids at the Kcr-interacting site, which subsequently could react with Kcr sites in a unique NAD⁺ -dependent manner. The covalent binder has been used to selectively recognize crotonylated proteins in extracted histone samples and in fixed cells.