Site-specific photo-crosslinking/cleavage for protein-protein interface identification reveals oligomeric assembly of lysosomal-associated membrane protein type 2A in mammalian cells.

ABSTRACT: Genetic code expansion enables site-specific photo-crosslinking 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 crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (N^ε -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-termin

SUBMITTER: Terasawa K 

PROVIDER: S-EPMC10659947 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature