Analyzing protein intermediate interactions in living E. coli cells using site-specific photo-crosslinking combined with chemical crosslinking

Miyazaki, Ryoji; Akiyama, Yoshinori

doi:10.1016/j.xpro.2023.102178

Cited by 1 publication

(1 citation statement)

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“…When p Bpa is incorporated at several sites in a protein and some sites allow crosslink formation, these sites are safely assumed to be near the binding interface. However, it is more difficult to identify the amino acid residues in the other protein that are crosslinked with p Bpa (Miyazaki & Akiyama, 2023 ). For example, the interaction between the periplasmic Zn‐metallopeptidase BepA and the lipopolysaccharide translocon LptD was demonstrated by firstly introducing p Bpa to BepA, and then conversely introducing p Bpa to LptD (Miyazaki et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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

Terasawa,

Seike,

Sakamoto

et al. 2023

Protein Science

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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 (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 within the target protein. A series of AllocLys‐OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal‐associated membrane protein type 2A (LAMP2A), a receptor of chaperone‐mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site‐specific crosslinking and site‐specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries.This article is protected by copyright. All rights reserved.

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Section: Introductionmentioning

confidence: 99%