Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.
Curr Protoc
; 4(8): e1103, 2024 Aug.
Article
in En
| MEDLINE
| ID: mdl-39105689
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Cross-Linking Reagents
Limits:
Animals
/
Humans
Language:
En
Journal:
Curr Protoc
Year:
2024
Document type:
Article
Affiliation country:
Japón
Country of publication:
Estados Unidos