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

Jianfei Feng, Pablo Martin-Baniandres, Michael J. Booth, Gianluca Veggiani, Mark Howarth, Hagan Bayley, David Rodriguez-Larrea
2020 Communications Biology  
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
more » ... ini 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.
doi:10.1038/s42003-020-0840-5 pmid:32246060 fatcat:ro2n5a4msbf5vl4x5tq3y4szvy