Fig. 2: Formation of an oligo(dC)₃₀-V5-oligo(dA)₃₀/αHL rotaxane.

a Prevention of release into the trans compartment. Oligo(dC)₃₀-V5-oligo(dA)₃₀ (biotinylated oligonucleotides) was treated with a sub-stoichiometric amount of mSA. A complex with one of the two oligonucleotides bound to mSA threads through the pore at +140 mV with the C terminus free oligonucleotide first. After unfolding and translocation of the protein (Trx V5), the translocated substrate remains arrested in the pore by the mSA. Reversal of the voltage polarity (−40 mV) drives the complex back into the cis compartment (retro-translocation), where it is released to the solution. The pore is left at level 1* (the open state at a negative potential). b Ionic current signal corresponding to the events shown in ‘a’. Level 1, open αHL pore; level 2, oligonucleotide threaded into the pore; level 3, unfolding intermediate; level 4, translocation (largely diffusive) of the unfolded protein; level 5, second oligonucleotide threaded into the pore; level 6, retro-translocation; level 1*, open pore (−40 mV). c Formation of a transmembrane protein rotaxane. The steps described in ‘a’ were carried out with SA to the trans compartment. After the protein translocation, an approximately 5 min delay allowed SA to bind to the biotinylated oligonucleotide. Then, after a voltage step to −40 mV, the protein was retro-translocated but remained associated with the αHL pore as a rotaxane. After a delay, a subsequent step to +140 mV can be used to probe the folded state of the Trx V5 in the cis compartment. d Ionic current signal characteristic of rotaxane formation. Level 1, open αHL pore (+140 mV); level 2, oligonucleotide threaded into the pore; level 3, unfolding intermediate; level 4, translocation of the unfolded protein; level 5, second oligonucleotide threaded into the pore; level 6, retro-translocation; level 7, initial leading oligonucleotide threaded into the pore. During level 7 (here 0.49 s) the protein refolds in the cis compartment and when the potential is stepped back to +140 mV, levels 2′, 3′, 4′, and 5′ appear. The prime symbol signifies that the levels are generated not from the native Trx, but from a refolded form.