Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions.
Nat Commun
; 8(1): 2240, 2017 12 21.
Article
em En
| MEDLINE
| ID: mdl-29269770
ABSTRACT
Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Reagentes de Ligações Cruzadas
/
Proteínas de Escherichia coli
/
Escherichia coli
/
Mapas de Interação de Proteínas
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article