Fig. 1: Differential tagging and co-translocational unfolding of Trx V5.

a Synthesis of oligo(dC)₃₀-V5-oligo(dA)₃₀. After each of the three steps, the product was purified and characterized by SDS-PAGE and MS (Supplementary Figs. 1–3). b An electric potential across a lipid bilayer containing a single αHL pore (cis at ground). In response to an applied positive potential, anions and cations are driven through the pore, which is recorded as a positive current. c When oligo(dC)₃₀-V5-oligo(dA)₃₀ is added to the cis compartment at +140 mV, one of the negatively-charged oligonucleotides is pulled into the αHL pore (level 2). The electrophoretic force acting on the oligonucleotide partly unfolds the protein (level 2→level 3). A relatively long-lived intermediate is generated in which the terminus of the polypeptide chain threaded through the pore (level 3). At this point, the remaining folded portion of Trx is unstable and, after a waiting time (level 3→level 4), it spontaneously unfolds and diffuses through the pore (level 4). d Electrical recording of a single translocation event showing levels 1–4. e Oligo(dC)₃₀-V5-oligo(dA)₃₀ can thread either N or C terminus-first. f Ionic current signals of level 2 for entry with the N terminus-first (blue) and C terminus-first (red). g Event histogram of the residual currents (I_res%) in level 2 during multiple translocation events of oligo(dC)₃₀-V5-oligo(dA)₃₀ through a single αHL pore showing two populations. The I_res% in level 2 was used to define the direction of translocation. The result was confirmed with two additional pores.