Multienzyme Catalysis in Phase-Separated Protein Condensates.
Cascade catalysis
Enzyme
Liquid–liquid phase separation
Protein condensate
Protein–protein interaction
Terpene biosynthesis
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
10
6
2022
pubmed:
11
6
2022
medline:
15
6
2022
Statut:
ppublish
Résumé
Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, and understanding how phase separation regulates multienzyme catalysis may need the help of in vitro investigations. Recently we have constructed synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. Here, we describe the methods for checking the enzyme assembly using fluorescent microscopy and centrifugation assay. We further provide steps for analysis of the cascade enzyme catalytic efficiencies inside the condensates, using enzymes from terpene biosynthesis pathway.
Identifiants
pubmed: 35687245
doi: 10.1007/978-1-0716-2269-8_20
doi:
Substances chimiques
Multienzyme Complexes
0
Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
345-354Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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