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

Curr Protoc. 2024 Aug;4(8):e1103. doi: 10.1002/cpz1.1103.

Abstract

Identification 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 sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

Keywords: genetic code expansion; protein‐protein interface; site‐specific cleavage; site‐specific crosslinking.

MeSH terms

  • Animals
  • Cross-Linking Reagents* / chemistry
  • Humans
  • Photochemical Processes
  • Protein Binding
  • Protein Interaction Mapping / methods
  • Proteins / chemistry
  • Proteins / metabolism

Substances

  • Cross-Linking Reagents
  • Proteins