Abstract
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.
Highlights
Understanding protein folding under conditions similar to those found in vivo remains challenging
The product, a maleimide-CBT crosslinker, was reacted with the 5′-thiol of a 3′-biotinylated oligonucleotide composed of 30 cytosines, and the resultant 5′-CBToligo(dC)30-biotin-3′ was purified by ion-exchange chromatography (Fig. 1a and Supplementary Fig. 1c–f)
The conjugate was reacted at the C-terminal cysteine with the 5′-thiol of a 3′-biotinylated oligonucleotide, composed of 30 adenines, that had been activated with 2,2′-dipyridyl disulfide (Supplementary Fig. 3b, c)[34]
Summary
Understanding protein folding under conditions similar to those found in vivo remains challenging. During or after synthesis by ribosomes, proteins may be transported between compartments in the cell[11] or into the extracellular medium[12] During these processes, they must cross membranes by using one of a diverse set of translocons. By using a mutant of DsbA, a disulfidebond-forming enzyme, the formation of covalent mixed-disulfide complexes between DsbA and a polypeptide substrate was slowed, which allowed the resolution of intermediates in the folding process. This approach is limited to disulfide-containing substrates in bacteria
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