Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates.
Commun Biol
; 3(1): 159, 2020 04 03.
Article
en En
| MEDLINE
| ID: mdl-32246060
Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or from a membrane translocon. Protein folding during membrane translocation is particularly difficult to study. Here, we describe a single-molecule method to characterize the folded state of individual proteins after membrane translocation, by monitoring the ionic current passing through the pore. We tag both N and C termini of a model protein, thioredoxin, with biotinylated oligonucleotides. Under an electric potential, one of the oligonucleotides is pulled through a α-hemolysin nanopore driving the unfolding and translocation of the protein. We trap the protein in the nanopore as a rotaxane-like complex using streptavidin stoppers. The protein is subjected to cycles of unfolding-translocation-refolding switching the voltage polarity. We find that the refolding pathway after translocation is slower than in bulk solution due to the existence of kinetic traps.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tiorredoxinas
/
Toxinas Bacterianas
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Membrana Celular
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Proteínas de Escherichia coli
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Rotaxanos
/
Proteínas Hemolisinas
Idioma:
En
Revista:
Commun Biol
Año:
2020
Tipo del documento:
Article
Pais de publicación:
Reino Unido