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Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates.

Communications biology (2020-04-05)
Jianfei Feng, Pablo Martin-Baniandres, Michael J Booth, Gianluca Veggiani, Mark Howarth, Hagan Bayley, David Rodriguez-Larrea
RÉSUMÉ

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.

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6-Amino-2-cyanobenzothiazole, 97%
Sigma-Aldrich
4-(Maleinimido)phenyl isocyanate, purum, ≥97.0% (CHN)