Protein‐Protein Interface Identification by Site‐Specific Photo‐Cross‐linking/Cleavage in Mammalian Cells

Abstract

AbstractIdentification of protein‐protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site‐specific photo‐cross‐linking by introducing photo‐reactive non‐canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein‐protein interactions and is applicable in mammalian cells. However, the identification of the cross‐linked region still remains challenging. Our new protocol enables its identification by pre‐installing a site‐specific cleavage site, an α‐hydroxy acid (Nε‐allyloxycarbonyl‐α‐hydroxyl‐L‐lysine acid, AllocLys‐OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α‐hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N‐terminus or C‐terminus, the crosslinked site is located on within the target protein. A series of AllocLys‐OH introductions narrows down the crosslinked region. This combination of site‐specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Search for crosslinkable sitesBasic Protocol 2: Site‐specific photo‐cross‐linking/cleavage